Bispecific cxcr4-cd4 polypeptides with potent anti-hiv activity

ABSTRACT

The present invention relates to bispecific polypeptides that are directed against the cellular receptor CD4 as well as a cellular co-receptor for HIV. Said polypeptides may be used to prevent human cell entry of HIV.

FIELD OF THE INVENTION

The present invention relates to bispecific polypeptides that aredirected against the cellular receptor CD4 as well as the cellularco-receptor for HIV. Said polypeptides may be used to prevent human cellentry of HIV.

BACKGROUND

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of common general knowledge in the field.

Infection with the Human Immunodeficiency Virus (HIV), if leftuntreated, almost always leads to death of the infected person. HIVinfects the CD4⁺ T-cells and leads to a decline in the number of CD4⁺T-cells in the infected person. When CD4⁺ T-cell numbers decline below acritical level, cell-mediated immunity is effectively lost, andinfections with a variety of opportunistic microbes appear, resulting inAcquired Immunodeficiency Syndrome (AIDS). Because the HIV-infectedperson can no longer defend against these opportunistic infections, thepatient will ultimately succumb to one of these infections.

Infection by HIV is mediated by the envelope glycoprotein (Env). Envforms a heterotrimer composed of the receptor binding subunit gp120 andthe HIV membrane anchored fusion protein subunit gp41 (see FIG. 1).Entry into host cells is mediated by gp120 interaction with CD4 thattriggers a conformational change allowing subsequent interaction with acellular co-receptor, principally CCR5 or CXCR4 (Dalgleish et al. (1984)Nature 312: 763-767; Klatzmann et al. (1984) Nature 312: 767-768; Mooreet al. (1997) Curr Opin Immunol 9: 551-562; Clapham & McKnight (2002) JGen Virol 83: 1809-1829). CCR5 is the predominant co-receptor used, butan altered use of CCR5 is selected for during progressive HIV infection.Association of gp120 with the co-receptor induces additionalconformational changes in gp41, which in turn promote mixing of themembrane lipids, ultimately facilitating fusion of the viral andcellular membranes (Weissenhorn et al. (1997) Nature 387: 426-430; Chanet al. (1997) Cell 89: 263-273; Weissenhorn et al. (2007) FEBS Lett 581:2150-2155). Once the virus has entered the T-cells, the virus hijacksthe replication machinery of the T-cell to produce additional copies ofHIV thereby furthering the infection.

Currently there is no cure available for HIV/AIDS. However, HIV infectedpersons can suppress replication of the virus through a variety ofanti-viral treatment options. Current treatment for HIV infectionconsists of anti-retroviral therapy, or ART. ART consists of theadministration of a cocktail of multiple anti-viral compounds. However,because HIV readily mutates the virus often becomes resistant to one ormore compounds in the ART cocktail. In addition, ART is associated witha number of side effects. While anti-retroviral adherence is the secondstrongest predictor of progression to AIDS and death, after CD4 count,incomplete adherence to ART is common in all groups of treatedindividuals. The average rate of adherence to ART is approximately 70%,despite the fact that long-term viral suppression requires near-perfectadherence. The resulting virologic treatment failure diminishes thepotential for long-term clinical success and increases the risk of drugresistance.

New therapies to treat HIV infection are needed therefore.

Next to the small-molecule anti-HIV compounds, neutralizing antibodieshave been engineered and tested. However, antibodies are necessarilydirected against the exterior of the virus. Indeed, Env is a main targetfor entry inhibitors (Matthews et al. (2004) Nat Rev Drug Discov 3:215-225) and most neutralizing antibodies are directed against gp120 orgp41 (Sattentau Q (2008) Curr Opin HIV AIDS 3: 368-374). Also in thiscase a crucial problem in HIV vaccine research is the generation ofcross-subtype neutralizing antibodies, which is due to the fact that HIVemploys a number of strategies to evade the immune response. Thisincludes highly variable gp120 regions, a carbohydrate shield (Wyatt etal. (1998) Nature 393: 705-711) and conformational masking of thereceptor binding site (Kwong et al. (2002) Nature 420: 678-682).

Nanobodies directed against various HIV-1 proteins have been described(Hinz et al. 2010 PLoS ONE 5:e10482; McCoy et al. 2014 Retrovirology11:83; Vercruysse et al. 2010 JBC 285:21768-21780; Bouchet et al., 2011Blood 117:3559-3568). However, various Nanobodies were directed againstintra-cellular HIV proteins, necessitating intracellular expression ofthe Nanobody. Moreover, in none of the cases, the development ofresistance by HIV against the Nanobody has been addressed.

Interestingly, the options for HIV to “mutate around” therapies directedat blocking cell entry appear to be more limited. Indeed, in a studyusing ibalizumab, an anti-CD4 monoclonal antibody, resistance wasdeveloped but the resistant isolates remained dependent on CD4 for viralentry, suggesting that resistance did not develop through the use ofalternative receptors (cf. Bruno & Jacobson 2010 J Antimicrob Chemother65:1839-1841). Nevertheless, also in this case resistance againstibalizumab developed eventually (cf. Fessel et al., 2011 Antiviral Res92:484-487).

PRO140 is a fully humanized IgG4 monoclonal antibody directed againstthe co-receptor CCR5. PRO140 blocks the HIV R5 subtype entry intoT-cells by masking the required co-receptor CCR5. In short term studies,resistance against PRO140 has not been observed. However, the potentialdevelopment of resistance in long term studies has not been addressed.PRO140 does not prevent the usage of the CXCR4 co-receptor. Forinstance, in up to 40 to 50% of individuals infected with B-HIV,progression to late stages of infection is associated with a switch inco-receptor specificity, with emergence of X4 (CXCR4) or R5X4(CCR5/CXCR4) viral variants (Bjorndal et al. J Virol 1997,71(10):7478-7487; Connor et al. J Exp Med 1997, 185(4):621-628). Theemergence of CXCR4-using HIV viruses is associated with rapid CD4⁺T-cell decline and progression from chronic to advanced stages of HIVinfection.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art, or to provide a usefulalternative.

The inventors demonstrated that binding by a bispecific polypeptidedirected against a co-receptor (CR) and the receptor CD4 resulted in asynergy of the two binding moieties against HIV infection (see Example4). Surprisingly, bispecific polypeptides were more effective than thecombination of the two individual moieties (see Example 8).

The combination of moieties in the bispecific polypeptide withnon-overlapping effects, i.e., a first moiety is directed against CD4and the second moiety is directed against a co-receptor, allows theapplication of more effective binders without increasing the overallnegative toxic effects to the host beyond unacceptable limits.

The inventors further demonstrated that accomplishing resistance by HIVagainst the bispecific polypeptide is extremely difficult, even in aforced laboratory setting (see Example 8). On the other hand, it wassurprisingly observed that even in a HIV strain made resistant againstone receptor, e.g. the anti-CD4 moiety, the bispecific polypeptide wasstill efficacious. Hence, this property expands the use of a bispecificpolypeptide to a possible efficacy against heterogeneous strains notinherently resistant to one moiety agent and another HIV strain notinherently resistant against another moiety.

Accordingly, the present invention relates to a polypeptide comprising afirst and a second immunoglobulin single variable domain (ISV), whereinsaid first ISV binds to CD4 and polymorphic variants present on thesurface of a cell; said second ISV binds to a co-receptor (CR) presenton the surface of said cell, preferably wherein said CR is chosen fromthe group consisting of CXCR4, CCR5, CCR1, CCR2, CCR3, CCR8, CX3CR1,CXCR6, FPRL1, GPR1, GPR15, APJ, and D6 and related polymorphic variants,preferably CXCR4, preferably human CR, preferably human CXCR4; andwherein said CR is not CD4, preferably said polypeptide inhibitsinfection of human immunodeficiency virus (HIV) or simianimmunodeficiency virus (SIV), more preferably said HIV is chosen fromthe group consisting of HIV-1 and HIV-2 (preferably HIV-1, preferablysubtype C), and preferably said cell is a human cell, preferably a humanCD4⁺-cell, even more preferably a human CD4⁺ T-cell.

Accordingly, the present invention relates to a polypeptide as describedsupra, wherein the average EC₅₀ value of HIV inhibition is of between 10nM and 0.1 pM, such as at an average EC₅₀ value of 10 nM or less, evenmore preferably at an average EC₅₀ value of 9 nM or less, such as lessthan 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as less than 400,300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, oreven less such as less than 0.4 pM; and/or wherein the IC₅₀ of HIVinhibition is lower than 50 nM, lower than 10 nM, lower than 1 nM orless, such as less than 0.5 nM or even less, such as less than 400, 300,200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, or evenless such as less than 0.4 pM; and/or wherein said polypeptide inhibitsHIV infection by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% andpreferably 95% or more, such as 100% (as measured in a HIV infectionassay); and/or wherein said polypeptide inhibits HIV fusion with CD4CXCR4⁺ cells; and/or wherein said polypeptide has an on rate constant(Kon) to said CD4 selected from the group consisting of at least about10² M⁻¹ s⁻¹, at least about 10³ M⁻¹ s⁻¹, at least about 10⁴ M⁻¹ s⁻¹, atleast about 10⁵ M⁻¹ s⁻¹, at least about 10⁶ M⁻¹ s⁻¹, 107 M⁻¹ s⁻¹, atleast about 10⁸ M⁻¹ s⁻¹, at least about 10⁹ M⁻¹ s⁻¹, and at least about10¹⁰ M⁻¹ s⁻¹, preferably as measured by surface plasmon resonance;and/or wherein said polypeptide has an off rate constant (Koff) to saidCD4 selected from the group consisting of at most about 10⁻³ s⁻, at mostabout 10⁻⁴ s⁻¹, at most about 10⁻⁵ s⁻¹, at most about 10⁻⁶ s⁻¹, at mostabout 10⁻⁷ s⁻¹, at most about 10⁻⁸ s⁻¹, at most about 10⁻⁹ s⁻¹, and atmost about 10⁻¹⁰ s⁻¹, preferably as measured by surface plasmonresonance; and/or wherein said polypeptide has a dissociation constant(K_(D)) to said CD4 selected from the group consisting of: at most about10⁻⁷ M, at most about 10⁻⁸ M, at most about 10⁻⁹ M, at most about 10⁻¹⁰M, at most about 10⁻¹¹ M, and at most about 10⁻¹² M, preferably asmeasured by surface plasmon resonance.

The present invention relates also to a polypeptide as described herein,wherein said first ISV binds to CD4 with an average KD value of between10 nM and 0.1 pM, such as at an average KD value of 10 nM or less, evenmore preferably at an average KD value of 9 nM or less, such as lessthan 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as less than 400,300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, oreven less such as less than 0.4 pM, preferably measured by SPR, forinstance as determined by a KinExA; and/or said polypeptide inhibitsmultimerisation by CD4 by less than about 50%, such as 40%, 30%, or 20%or even less than 10%, such as less than 5%; and/or said polypeptideinhibits recruiting Lck by CD4 by less than about 50%, such as 40%, 30%,or 20% or even less than 10%, such as less than 5%.

The present invention relates also to a polypeptide as described herein,wherein said polypeptide has an on rate constant (Kon) to said CRselected from the group consisting of at least about 10² M⁻¹ s⁻¹, atleast about 10³ M⁻¹ s⁻¹, at least about 10⁴ M⁻¹ s⁻¹, at least about 10⁵M⁻¹ s⁻¹, at least about 10⁶ M⁻¹ s⁻¹, 10⁷ M⁻¹ s⁻¹, at least about 10⁸ M⁻¹s⁻¹, at least about 10⁹ M⁻¹ s⁻¹, and at least about 10¹⁰ M⁻¹ s⁻¹,preferably as measured by surface plasmon resonance, said CR ispreferably CXCR4; and/or wherein said polypeptide has an off rateconstant (Koff) to said CR selected from the group consisting of at mostabout 10⁻³ s⁻¹, at most about 10⁻⁴ s⁻¹, at most about 10⁻⁵ s⁻¹, at mostabout 10⁻⁶ s⁻¹, at most about 10⁻⁷ s⁻¹, at most about 10⁸ s⁻¹, at mostabout 10⁻⁹ s⁻¹, and at most about 10⁻¹⁰ s⁻¹, preferably as measured bysurface plasmon resonance, said CR is preferably CXCR4; and/or saidpolypeptide has a dissociation constant (K_(D)) to said CR selected fromthe group consisting of: at most about 10⁻⁷ M, at most about 10⁻⁸ M, atmost about 10⁻⁹ M, at most about 10⁻¹⁰ M, at most about 10⁻¹¹ M, and atmost about 10⁻¹²M, preferably as measured by surface plasmon resonance,said CR is preferably CXCR4; and/or said second ISV binds to said CRwith an average KD value of between 10 nM and 0.1 pM, such as at anaverage KD value of 10 nM or less, even more preferably at an average KDvalue of 9 nM or less, such as less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nMor even less, such as less than 400, 300, 200, 100, 50, 40, 30, 20, 10,9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, or even less such as less than 0.4pM, preferably measured by SPR, for instance as determined by a KinExA,said CR is preferably CXCR4; and/or said polypeptide inhibits binding ofa natural ligand to said CR by less than about 50%, such as 40%, 30%, or20% or even less than 10%, such as less than 5%.

The present invention relates also to a polypeptide as described herein,wherein said CR is preferably CXCR4, and preferably the natural ligandis Stromal Cell-Derived Factor-1 beta (SDF-1β) or Stromal Cell-DerivedFactor-1 alpha (SDF-1α); and preferably the IC₅₀ of SDF-1a or SDF-1βdisplacement from CXCR4 in the presence of the polypeptide is 10 nM orhigher, 250 nM or higher, or 1 μM or higher; and even more preferablythe IC₅₀ of SDF-1a or SDF-1β displacement from CXCR4 in the presence ofthe polypeptide is greater than the IC₅₀ of HIV inhibition.

The present invention relates also to a polypeptide as described herein,further comprising a serum protein binding moiety, preferably bindingserum albumin, a non-antibody based polypeptide or PEG.

Preferably said serum protein binding moiety is an immunoglobulin singlevariable domain binding serum albumin More preferably, said ISV bindingserum albumin essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 is SEQ ID NO: 124, CDR2 is SEQ ID NO: 125CDR3 is SEQ ID NO: 126, such as chosen from the group consisting ofAlb8, Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A,Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG.

The present invention relates also to a polypeptide as described herein,wherein said first ISV and said second ISV and possibly said ISV bindingserum albumin are directly linked to each other or are linked via alinker; preferably chosen from the group consisting of linkers of 5GS,7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS.

The present invention relates also to a polypeptide as described herein,wherein said ISV is a Nanobody®, a V_(HH), a humanized V_(HH) or acamelized V_(H).

The present invention relates also to a polypeptide as described herein,wherein said first ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which (i) CDR1 is chosen from the groupconsisting of SEQ ID NOs: 82-85; and amino acid sequences that have 1, 2or 3 amino acid difference(s) with SEQ ID NOs: 82-85; (ii) CDR2 ischosen from the group consisting of SEQ ID NOs: 88-91; and amino acidsequences that have 1, 2 or 3 amino acid difference(s) with SEQ ID NOs:88-91; and (iii) CDR3 is chosen from the group consisting of SEQ ID NO:96-99 and amino acid sequences that have 1, 2, 3 or 4 amino aciddifference(s) with SEQ ID NOs: 96-99; preferably CDR1 is SEQ ID NO: 85,CDR2 is SEQ ID NO: 91 and CDR3 is SEQ ID NO: 99. Also, the presentinvention relates also to a polypeptide as described supra, wherein saidfirst ISV is chosen from the group consisting of 01B6 (SEQ ID NO: 17),01E2 (SEQ ID NO: 18), 01H12 (SEQ ID NO: 19) and 03F11 (SEQ ID NO: 20),preferably said first ISV is clone 03F11 (SEQ ID NO: 20).

The present invention relates also to a polypeptide as described herein,wherein said second ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which (i) CDR1 is chosen from the groupconsisting of SEQ ID NOs: 34-40 and amino acid sequences that have 1, 2or 3 amino acid difference(s) with SEQ ID NOs: 34-40; (ii) CDR2 ischosen from the group consisting of SEQ ID NOs: 48-56; and amino acidsequences that have 1, 2 or 3 amino acid difference(s) with SEQ ID NOs:48-56; and (iii) CDR3 is chosen from the group consisting of SEQ ID NO:67-75 and amino acid sequences that have 1, 2, 3 or 4 amino aciddifference(s) with SEQ ID NOs: 67-75; preferably CDR1 is SEQ ID NO: 35,CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 69. Also, the presentinvention relates also to a polypeptide as described supra, wherein saidsecond ISV is chosen from the group consisting of 238D4 (SEQ ID NO: 4),281A5 (SEQ ID NO: 5), 281E10 (SEQ ID NO: 6), 281D4 (SEQ ID NO: 7), 281A6(SEQ ID NO: 8), 281F12 (SEQ ID NO: 9), 283B6 (SEQ ID NO: 10), 283E2 (SEQID NO: 11), 283F1 (SEQ ID NO: 12), 15F5 (SEQ ID NO: 13), 15G11 (SEQ IDNO: 14), 15A1 (SEQ ID NO: 15) and 10C3 (SEQ ID NO: 16), preferably inwhich said second ISV is 281F12 (SEQ ID NO: 9).

Also, the present invention relates to a polypeptide as describedherein, wherein said first ISV is chosen from the group consisting of01B6 (SEQ ID NO: 17), 01E2 (SEQ ID NO: 18), 01H12 (SEQ ID NO: 19) and03F11 (SEQ ID NO: 20), and wherein said second ISV is chosen from thegroup consisting of 238D4 (SEQ ID NO: 4), 281A5 (SEQ ID NO: 5), 281E10(SEQ ID NO: 6), 281D4 (SEQ ID NO: 7), 281A6 (SEQ ID NO: 8), 281F12 (SEQID NO: 9), 283B6 (SEQ ID NO: 10), 283E2 (SEQ ID NO: 11), 283F1 (SEQ IDNO: 12), 15F5 (SEQ ID NO: 13), 15G11 (SEQ ID NO: 14), 15A1 (SEQ ID NO:15) and 10C3 (SEQ ID NO: 16), preferably said polypeptide is chosen fromthe group consisting of 03F11-9GS-281F12 (SEQ ID NO: 101),03F11-25GS-281F12 (SEQ ID NO: 102), 03F11-35GS-281F12 (SEQ ID NO: 103),281F12-9GS-03F11 (SEQ ID NO: 104), 281F12-25GS-03F11 (SEQ ID NO: 105),281F12-35GS-03F11 (SEQ ID NO: 106),15G11(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 107),15F05(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 108), and281F12(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 109).

In addition, the present invention relates also to a polypeptide asdescribed supra, for use in treating a subject in need thereof (infectedwith HIV, HIV-1, subtype C). Also, the present invention relates also toa (pharmaceutical) composition comprising the polypeptide as describedsupra.

The present invention relates also to a method for delivering aprophylactic or therapeutic polypeptide to a specific location, tissueor cell type in the body, the method comprising the steps ofadministering to a subject a polypeptide as described supra.

The present invention relates also to a method for treating a subject inneed thereof comprising administering a polypeptide as described herein,preferably wherein said subject is infected with HIV R5, HIV X4, and/orHIV X4R5.

The present invention relates also to a method for treating a subjectinfected with HIV, comprising administering a polypeptide as describedherein, wherein said HIV infected subject does not develop resistance tosaid polypeptide for at least 6 months, etc., preferably, in acombination treatment with PR, RTI and/or NRTI.

The present invention relates also to a method for treating a subjectinfected with HIV, comprising administering a polypeptide as describedherein, wherein said subject is resistant against at least one otheranti-HIV agent, such as to one or more protease inhibitors (PRs), e.g.amprenavir (AMP), atazanavir (ATV), indinavir (IDV), lopinavir (LPV),nelfinavir (NFV), ritonavir (RTV) or saquinavir (SQV); and/or reversetranscriptase inhibitors (RTIs), e.g. a non-nucleoside reversetranscriptase inhibitor (NNRTI) [abacavir (ABC), delavirdine (DLV),efavirenz (EFV), nevirapine (NVP) and tenofovir (TFV)]; or a nucleosideanalogue reverse transcriptase inhibitor (NRTI) [didanosine (ddl),stavudine (d4T), lamivudine (3TC) and zidovudine (ZDV)].

The present invention relates also to a method for lowering theHIV-titer in a subject, the method comprising administering to thesubject a therapeutically effective amount of a polypeptide as describedherein to lower the HIV-titer in the subject.

The present invention relates also to a method of treating a symptom ofacquired immune deficiency syndrome in a human subject infected with HIVthat is, or has become, resistant to a non-antibody CD4 and/or CRantagonist, comprising administering to the human subject a polypeptideas described herein in an amount effective to treat the symptom ofacquired immune deficiency syndrome in the human subject.

The present invention relates also to a method for preventing HIVinfection in a subject, the method comprising administering to thesubject a therapeutically effective amount of a polypeptide as describedherein to prevent infection of the subject by HIV.

The present invention relates also to a method of inhibiting HIVinfection of a susceptible cell in a subject by an HIV virus that isresistant, or has become resistant, to a CD4 antagonist, which comprisessubjecting the susceptible cell to an effective HIV infection inhibitingdose of a polypeptide as described herein (which inhibits HIV fusionwith CD4+CXCR4+ cells), preferably wherein the effective HIV infectioninhibiting dose comprises from 0.1 mg per kg to 25 mg per kg of thesubject's body weight, so as to thereby inhibit the infection of thesusceptible cell by HIV1 that is resistant, or has become resistant, tothe CD4 antagonist.

The present invention relates also to a polypeptide comprising a firstand a second immunoglobulin single variable domain (ISV), wherein saidfirst ISV binds to CD4 present on the surface of a cell; said second ISVbinds to CXCR4 present on the surface of said cell; and wherein saidfirst ISV essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which

-   (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 85, 84,    83 and 82; and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 85, 84, 83 and 82;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 91, 90,    89 and 88; and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 91, 90, 89 and 88; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 99, 98,    97 and 96; and amino acid sequences that have 1, 2, 3 or 4 amino    acid difference(s) with SEQ ID NOs: 99, 98, 97 and 96;    and, wherein said second ISV essentially consists of 4 framework    regions (FR1 to FR4, respectively) and 3 complementarity determining    regions (CDR1 to CDR3, respectively), in which-   (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 35, 34,    36-40; and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 35, 34, 36-40;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 50,    48-49 and 51-56; and amino acid sequences that have 1, 2 or 3 amino    acid difference(s) with SEQ ID NOs: 50, 48-49 and 51-56; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 69,    67-68, 70-75 and amino acid sequences that have 1, 2, 3 or 4 amino    acid difference(s) with SEQ ID NOs: 69, 67-68, 70-75.

The present invention relates also to a polypeptide as described herein,wherein said first ISV is chosen from the group consisting of 03F11 (SEQID NO: 20), 01B6 (SEQ ID NO: 17), 01E2 (SEQ ID NO: 18), and 01H12 (SEQID NO: 19), and wherein said second ISV is chosen from the groupconsisting of 281F12 (SEQ ID NO: 9), 238D4 (SEQ ID NO: 4), 281A5 (SEQ IDNO: 5), 281E10 (SEQ ID NO: 6), 281D4 (SEQ ID NO: 7), 281A6 (SEQ ID NO:8), 283B6 (SEQ ID NO: 10), 283E2 (SEQ ID NO: 11), 283F1 (SEQ ID NO: 12),15F5 (SEQ ID NO: 13), 15G11 (SEQ ID NO: 14), 15A1 (SEQ ID NO: 15) and10C3 (SEQ ID NO: 16).

The present invention relates also to a polypeptide as described herein,wherein said polypeptide prevents infection of said HIV for at least 3months, such as at least 6 months, or even longer such as e.g. 9 m, 11m, 1 y, 1.5 y, 2 y or even longer.

The present invention relates also to a polypeptide as described herein,wherein said polypeptide inhibits binding of a natural ligand to saidCXCR4 by less than about 50%, such as 40%, 30%, or 20% or even less than10%, such as less than 5%, wherein the natural ligand is StromalCell-Derived Factor-1 beta (SDF-13) or Stromal Cell-Derived Factor-1alpha (SDF-1a).

The present invention relates also to a polypeptide as described herein,for use in treating and/or preventing HIV infection in a subject.Preferably, said polypeptide prevents HIV infection for at least 3months, such as at least 6 months, or even longer such as e.g. 9 m, 11m, 1 y, 1.5 y, 2 y or even longer. The present invention relates also toa polypeptide as described herein, wherein said polypeptide inhibits HIVinfection by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably95% or more, such as 100%, for instance as measured in a HIV infectionassay. The present invention relates also to a polypeptide as describedherein, for use in treating and/or preventing HIV infection in asubject, wherein the polypeptide inhibits HIV fusion with CD4+CXCR4+cells. The present invention relates also to a polypeptide as describedherein, for use in treating and/or preventing HIV infection in asubject, wherein said polypeptide inhibits binding of a natural ligandto said CXCR4 by less than about 50%, such as 40%, 30%, or 20% or evenless than 10%, such as less than 5%, wherein the natural ligand isStromal Cell-Derived Factor-1 beta (SDF-13) or Stromal Cell-DerivedFactor-1 alpha (SDF-1a).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the model system.

FIGS. 2A-2D: Identification of human CD4-specific Nanobodies directedagainst the gp120 binding site. FIG. 2A: Phage binding to recombinanthuman CD4 in ELISA. FIG. 2B: Binding to cell-expressed CD4 on Jurkat andTHP-1 cells, but not Ba/F3 cells by flow cytometry, using detection ofthe myc-tag. FIG. 2C: Blockade of CD4-gp120 interaction in competitionELISA for selected CD4 Nanobodies. In FIGS. 2B and 2C anti-hCD4monoclonal mAb A-1 (Diaclone) was used as positive control. FIG. 2D:Binding of Nanobody 3F11 to human T cells.

FIG. 2.1: Radio-ligand displacement analysis of CXCR4 Nanobodies forbinding to human CXCR4. Membrane extracts of Hek293 cells transfectedwith CXCR4 were incubated with serial dilutions of purified Nanobodiesand 75 pM of [¹²⁵1]-CXCL12. Non-specific binding was determined inpresence of 100 nM cold SDF-1. Means of 3 experiments are shown.

FIG. 2.2: Binding of the monovalent and bispecific CD4-CXCR4 Nanobodiesto CXCR4 on viral lipoparticles (CXCR4-lip) versus empty controllipoparticles (null-lip) in ELISA. Bound Nanobodies were detected withmouse anti-c-myc and rabbit anti-Mouse-HRP antibodies.

FIG. 2.3: Binding affinity analysis of monovalent and bispecificCXCR4-CD4 polypeptides to cell lines that expresses both CXCR4 and CD4,Jurkat E6.1, THP-1 and MOLM-13 cells. Onset shows the relativeexpression levels of CD4 and CXCR4 on these cell lines, as determinedwith anti-CD4 mAb A-1 (Diaclone) and anti-CXCR4 mAb 12G5 (R&D systems)control antibodies. Bispecific polypeptides with the 35GS linker wereused. Nanobody detection was done via anti-tag antibodies.

FIG. 2.4: Inhibition of SDF-1 mediated chemotaxis of monovalent andCXCR4-CD4 bispecific polypeptides to Jurkat E6.1 and Molm-13 cells.Bispecific polypeptides with the 35GS-linkers are shown.

FIG. 3: Inhibition of anti-CXCR4 mAb 12G5 binding by monovalent andCXCR4-CD4 bispecific polypeptides to Jurkat E6.1 cells and THP-1 cells.Bound 12G5 antibody was detected with Goat anti-Mouse-PE (JacksonImmunoResearch).

FIG. 4: Dose-dependent inhibition of HIV-1 NL4.3 infectivity in MT-4cells by bispecific CXCR4-CD4 polypeptides in comparison with a mixtureof monovalent Nanobodies. Detection was done with the MTS viabilitystaining method.

FIG. 5: Inhibition of HIV1 infectivity by CXCR4-CD4 polypeptides withdifferent linker lengths of wild-type NL4.3 and AMD3100-resistant HIV-1variants in MT-4 cells. AMD-3100 was used as control compound. AverageIC₅₀ of three experiments is shown.

FIG. 6: Ranking of a large panel of CXCR4 Nanobodies for HIV-1neutralization capacity, assessed on NL4.1 infection in MT-4 cells.AMD-3100 was included as control compound. Average IC₅₀ values of twoexperiments is shown.

FIG. 7: Identification of CXCR4 Nanobodies directed against the gp120binding site on CXCR4 but not competing with the ligand. Panel A:Comparison of ligand competing and HIV-1 neutralisation affinities of aselection of CXCR4 Nanobodies. CXCR4 Nanobody 281F12 and AMD-3100 areincluded as references. Panel B: Ligand displacement analysis ofselected CXCR4 Nanobodies to CXCR4 expressed on Hek-293 cells.Biotinylated-SDF-1 (30 nM, EC₃₀ concentration) was used for detection.AMD-3100 and anti-CXCR4 mAb 12G5 are included as reference compounds.

FIG. 8: Inhibition of anti-CXCR4 mAb 12G5 and AMD-3100 binding to CXCR4by monovalent CXCR4 Nanobodies on Jurkat E6.1 cells by flow cytometry.Bound 12G5 antibody was detected with Goat anti-Mouse-PE (JacksonImmunoResearch). In case of AMD-3100 competition, Nanobodies were usedat the EC₃₀ concentrations and competed with increasing concentrationsof AMD-3100. Bound Nanobody was detected via anti-myc detection.

FIG. 9: Inhibition of SDF-1 mediated chemotaxis of half-life extendedCXCR4-CD4 bispecific polypeptides to Jurkat E6.1 cells (panel A) andMolm-13 cells (panel B). Chemotaxis was assessed to 750 pM SDF-1(Jurkat) or 1 nM SDF-1 (MOLM-13) over three hours.

FIG. 10: Binding analysis of monovalent anti-CD4 Nanobody 3F11 to CD4⁺cynomolgus HSC-F T cells by flow cytometry. Bound Nanobodies weredetected with mouse anti-Flag and Goat anti-Mouse-PE (JacksonImmunoResearch) antibodies.

DESCRIPTION OF THE INVENTION

The present invention relates to particular polypeptides, also referredto as “polypeptides of the invention” that comprise or essentiallyconsist of (i) a first building block consisting essentially of a firstimmunoglobulin single variable domain, wherein said first immunoglobulinsingle variable domain binds a first target, preferably an HIV receptor,such as CD4, on the surface of a cell; and (ii) a second building blockconsisting essentially of a second immunoglobulin single variabledomain, wherein said second immunoglobulin single variable domain bindsa second target, preferably an HIV co-receptor (CR), on the surface of acell, and wherein said CR is not CD4.

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020079.-   b) Unless indicated otherwise, the term “immunoglobulin single    variable domain” or “ISV” is used as a general term to include but    not limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H) or V_(L) domains, respectively. The terms    antigen-binding molecules or antigen-binding protein are used    interchangeably and include also the term Nanobodies. The    immunoglobulin single variable domains can be light chain variable    domain sequences (e.g., a V_(L)-sequence), or heavy chain variable    domain sequences (e.g., a V_(H)-sequence); more specifically, they    can be heavy chain variable domain sequences that are derived from a    conventional four-chain antibody or heavy chain variable domain    sequences that are derived from a heavy chain antibody. Accordingly,    the immunoglobulin single variable domains can be domain antibodies,    or immunoglobulin sequences that are suitable for use as domain    antibodies, single domain antibodies, or immunoglobulin sequences    that are suitable for use as single domain antibodies, “dAbs”, or    immunoglobulin sequences that are suitable for use as dAbs, or    Nanobodies, including but not limited to V_(HH) sequences, as well    as humanized V_(HH) sequences and camelized V_(H) sequences. The    invention includes immunoglobulin sequences of different origin,    comprising mouse, rat, rabbit, donkey, human and camelid    immunoglobulin sequences. The immunoglobulin single variable domain    includes fully human, humanized, otherwise sequence optimized or    chimeric immunoglobulin sequences. The immunoglobulin single    variable domain and structure of an immunoglobulin single variable    domain can be considered—without however being limited thereto—to be    comprised of four framework regions or “FR's”, which are referred to    in the art and herein as “Framework region 1” or “FR1”; as    “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and    as “Framework region 4” or “FR4”, respectively; which framework    regions are interrupted by three complementary determining regions    or “CDR's”, which are referred to in the art as “Complementarity    Determining Region 1” or “CDR1”; as “Complementarity Determining    Region 2” or “CDR2”; and as “Complementarity Determining Region 3”    or “CDR3”, respectively. It is noted that the terms Nanobody or    Nanobodies are registered trademarks of Ablynx N.V. and thus may    also be referred to as NANOBODY® or NANOBODIES®, respectively.-   c) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020079.-   d) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   e) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020079    of Ablynx N.V. entitled “Immunoglobulin single variable domains    directed against IL-6R and polypeptides comprising the same for the    treatment of diseases and disorders associated with IL-6 mediated    signalling”.-   f) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020079    (incorporated herein by reference).-   g) For the purposes of comparing two or more immunoglobulin single    variable domains or other amino acid sequences such e.g. the    polypeptides of the invention etc., the percentage of “sequence    identity” between a first amino acid sequence and a second amino    acid sequence (also referred to herein as “amino acid identity”) may    be calculated or determined as described in paragraph f) on pages 49    and 50 of WO 08/020079 (incorporated herein by reference), such as    by dividing [the number of amino acid residues in the first amino    acid sequence that are identical to the amino acid residues at the    corresponding positions in the second amino acid sequence] by [the    total number of amino acid residues in the first amino acid    sequence] and multiplying by [100%], in which each deletion,    insertion, substitution or addition of an amino acid residue in the    second amino acid sequence—compared to the first amino acid    sequence—is considered as a difference at a single amino acid    residue (position), i.e., as an “amino acid difference” as defined    herein; or using a suitable computer algorithm or technique, again    as described in paragraph f) on pages 49 and 50 of WO 08/020079    (incorporated herein by reference).    -   Also, in determining the degree of sequence identity between two        immunoglobulin single variable domains, the skilled person may        take into account so-called “conservative” amino acid        substitutions, as described on page 50 of WO 08/020079.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al., Principles of        Protein Structure, Springer-Verlag, 1978, on the analyses of        structure forming potentials developed by Chou and Fasman,        Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,        and on the analysis of hydrophobicity patterns in proteins        developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:        140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,        198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,        1986, all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        Nanobodies is given in the description herein and in the general        background art cited above. Also, for this purpose, the crystal        structure of a V_(HH) domain from a llama is for example given        by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803        (1996); Spinelli et al., Natural Structural Biology (1996); 3,        752-757; and Decanniere et al., Structure, Vol. 7, 4, 361        (1999). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   h) Immunoglobulin single variable domains and nucleic acid sequences    are said to be “exactly the same” if they have 100% sequence    identity (as defined herein) over their entire length.-   i) When comparing two immunoglobulin single variable domains, the    term “amino acid difference” refers to an insertion, deletion or    substitution of a single amino acid residue on a position of the    first sequence, compared to the second sequence; it being understood    that two immunoglobulin single variable domains can contain one, two    or more such amino acid differences.-   j) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020079. Unless the context    clearly requires otherwise, throughout the description and the    claims, the words “comprise”, “comprising”, and the like are to be    construed in an inclusive sense as opposed to an exclusive or    exhaustive sense; that is to say, in the sense of “including, but    not limited to”.-   k) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020079.-   l) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020079.-   m) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph l) on page 53 of WO 08/020079.-   n) As further described in paragraph m) on page 53 of WO 08/020079,    an amino acid sequence (such as an antibody, a polypeptide of the    invention, or generally an antigen binding protein or polypeptide or    a fragment thereof) that can (specifically) bind to, that has    affinity for and/or that has specificity for a specific antigenic    determinant, epitope, antigen or protein (or for at least one part,    fragment or epitope thereof) is said to be “against” or “directed    against” said antigenic determinant, epitope, antigen or protein.-   o) The term “specificity” refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as an ISV,    such as e.g. a Nanobody, or a polypeptide of the invention) molecule    can bind. The specificity of an antigen-binding protein can be    determined based on affinity and/or avidity.    -   The affinity, represented by the equilibrium constant for the        dissociation of an antigen with an antigen-binding protein        (K_(D) or KD), is a measure for the binding strength between an        antigenic determinant, i.e. the target, and an antigen-binding        site on the antigen-binding protein, i.e. the ISV or Nanobody:        the lesser the value of the K_(D), the stronger the binding        strength between an antigenic determinant and the        antigen-binding molecule (alternatively, the affinity can also        be expressed as the affinity constant (K_(A)), which is        1/K_(D)). As will be clear to the skilled person (for example on        the basis of the further disclosure herein), affinity can be        determined in a manner known per se, depending on the specific        antigen of interest.    -   Avidity is the affinity of the polypeptide, i.e. the ligand is        able to bind via two (or more) pharmacophores (ISV) in which the        multiple interactions synergize to enhance the “apparent”        affinity. Avidity is the measure of the strength of binding        between the polypeptide of the invention and the pertinent        antigens. The polypeptide of the invention is able to bind via        its two (or more) building blocks, such as ISVs or Nanobodies,        to the at least two targets, in which the multiple interactions,        e.g. the first building block, ISV or Nanobody binding to the        first target and the second building block, ISV, or Nanobody        binding to the second target, synergize to enhance the        “apparent” affinity. Avidity is related to both the affinity        between an antigenic determinant and its antigen binding site on        the antigen-binding molecule and the number of pertinent binding        sites present on the antigen-binding molecules. For example, and        without limitation, polypeptides that contain two or more        building blocks, such as ISVs or Nanobodies directed against        different targets on a cell and in particular against human        CXCR4 and human CD4 may (and usually will) bind with higher        avidity than each of the individual monomers or individual        building blocks, such as, for instance, the monovalent ISVs or        Nanobodies, comprised in the polypeptides of the invention.    -   In the present invention, monovalent antigen-binding proteins        (such as the building blocks, ISVs, amino acid sequences,        Nanobodies and/or polypeptides of the invention) are said to        bind to their antigen with a high affinity when the dissociation        constant (KD) is 10⁻⁹ to 10⁻¹² moles/liter or less, and        preferably 10⁻¹⁰ to 10⁻¹² moles/liter or less and more        preferably 1011 to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁹ to 10¹² liter/moles or more, and        preferably 10¹⁰ to 10¹² liter/moles or more and more preferably        10¹¹ to 10¹² liter/moles).    -   In the present invention, monovalent antigen-binding proteins        (such as the building blocks, ISVs, amino acid sequences,        Nanobodies and/or polypeptides of the invention) are said to        bind to their antigen with a low affinity when the dissociation        constant (K_(D)) is 10⁻⁶ to 10⁻⁹ moles/liter or more, and        preferably 10⁻⁶ to 10⁻⁸ moles/liter or more and more preferably        10⁻⁶ to 10⁻⁷ moles/liter (i.e. with an association constant        (K_(A)) of 10⁶ to 10⁹ liter/moles or more, and preferably 10⁶ to        10⁸ liter/moles or more and more preferably 10⁶ to 10⁷        liter/moles).    -   A medium affinity can be defined as values ranging in between        high-low, e.g. 10⁻¹⁰ to 10⁻⁸ moles/liter.    -   Any K_(D) value greater than 10⁻⁴ mol/liter (or any K_(A) value        lower than 10⁴ liter/mol) is generally considered to indicate        non-specific binding.    -   The polypeptides of the invention comprise a first and a second        building block, e.g. a first and a second ISV, or a first and a        second Nanobody. Preferably the affinity of each building block,        e.g. ISV or Nanobody, is determined individually. In other        words, the affinity is determined for the monovalent building        block, ISV or Nanobody, independent of avidity effects due to        the other building block, ISV or Nanobody, which might or might        not be present. The affinity for a monovalent building block,        ISV or Nanobody can be determined on the monovalent building        block, ISV or Nanobody per se, i.e. when said monovalent        building block, ISV or Nanobody is not comprised in the        polypeptide of the invention. In the alternative or in addition,        the affinity for a monovalent building block, ISV or Nanobody        can be determined on one target while the other target is        absent.    -   The binding of an antigen-binding protein to an antigen or        antigenic determinant can be determined in any suitable manner        known per se, including, for example, Scatchard analysis and/or        competitive binding assays, such as radioimmunoassays (RIA),        enzyme immunoassays (EIA) and sandwich competition assays, and        the different variants thereof known per se in the art; as well        as the other techniques mentioned herein.    -   The dissociation constant may be the actual or apparent        dissociation constant, as will be clear to the skilled person.        Methods for determining the dissociation constant will be clear        to the skilled person, and for example include the techniques        mentioned herein. In this respect, it will also be clear that it        may not be possible to measure dissociation constants of more        than 10⁻⁴ moles/liter or 10³ moles/liter (e.g., of 10⁻²        moles/liter). Optionally, as will also be clear to the skilled        person, the (actual or apparent) dissociation constant may be        calculated on the basis of the (actual or apparent) association        constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].    -   The affinity denotes the strength or stability of a molecular        interaction. The affinity is commonly given as by the K_(D), or        dissociation constant, which has units of mol/liter (or M). The        affinity can also be expressed as an association constant,        K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or        M⁻¹). In the present specification, the stability of the        interaction between two molecules (such as an amino acid        sequence, ISV, such as e.g. a Nanobody, or polypeptide of the        invention and its intended target) will mainly be expressed in        terms of the K_(D) value of their interaction; it being clear to        the skilled person that in view of the relation K_(A)=1/K_(D),        specifying the strength of molecular interaction by its K_(D)        value can also be used to calculate the corresponding K_(A)        value. The K_(D)-value characterizes the strength of a molecular        interaction also in a thermodynamic sense as it is related to        the free energy (DG) of binding by the well-known relation        DG=RT·ln(K_(D)) (equivalently DG=−RT·ln(K_(A))), where R equals        the gas constant, T equals the absolute temperature and ln        denotes the natural logarithm.    -   The K_(D) for biological interactions which are considered        meaningful (e.g. specific) are typically in the range of 10-10M        (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,        the lower is its K_(D).    -   The K_(D) can also be expressed as the ratio of the dissociation        rate constant of a complex, denoted as k_(off), to the rate of        its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)        and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹        (where s is the SI unit notation of second). The on-rate k_(on)        has units M⁻¹ s⁻¹. The on-rate may vary between 10² M⁻¹ s⁻¹ to        about 10⁷ M⁻¹ s⁻¹, approaching the diffusion-limited association        rate constant for bimolecular interactions. The off-rate is        related to the half-life of a given molecular interaction by the        relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between        10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple        days) to 1 s⁻¹ (t_(1/2)=0.69 s).    -   The affinity of a molecular interaction between two molecules        can be measured via different techniques known per se, such as        the well-known surface plasmon resonance (SPR) biosensor        technique (see for example Ober et al., Intern. Immunology, 13,        1551-1559, 2001). The term “surface plasmon resonance”, as used        herein, refers to an optical phenomenon that allows for the        analysis of real-time biospecific interactions by detection of        alterations in protein concentrations within a biosensor matrix,        where one molecule is immobilized on the biosensor chip and the        other molecule is passed over the immobilized molecule under        flow conditions yielding k_(on), k_(off) measurements and hence        K_(D) (or K_(A)) values. This can for example be performed using        the well-known BIACORE® system (BIAcore International AB, a GE        Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For        further descriptions, see Jonsson, U., et al. (1993) Ann. Biol.        Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques        11:620-627; Johnsson, B., et al. (1995) J Mol. Recognit. 8:        125-131; and Johnnson, B., et al. (1991) Anal. Biochem.        198:268-277.    -   It will also be clear to the skilled person that the measured        K_(D) may correspond to the apparent K_(D) if the measuring        process somehow influences the intrinsic binding affinity of the        implied molecules for example by artefacts related to the        coating on the biosensor of one molecule. Also, an apparent        K_(D) may be measured if one molecule contains more than one        recognition site for the other molecule. In such situation the        measured affinity may be affected by the avidity of the        interaction by the two molecules.    -   Another approach that may be used to assess affinity is the        2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of        Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This        method establishes a solution phase binding equilibrium        measurement and avoids possible artefacts relating to adsorption        of one of the molecules on a support such as plastic.    -   However, the accurate measurement of K_(D) may be quite        labour-intensive and as consequence, often apparent K_(D) values        are determined to assess the binding strength of two molecules.        It should be noted that as long all measurements are made in a        consistent way (e.g. keeping the assay conditions unchanged)        apparent K_(D) measurements can be used as an approximation of        the true K_(D) and hence in the present document K_(D) and        apparent K_(D) should be treated with equal importance or        relevance.    -   Finally, it should be noted that in many situations the        experienced scientist may judge it to be convenient to determine        the binding affinity relative to some reference molecule. For        example, to assess the binding strength between molecules A and        B, one may e.g. use a reference molecule C that is known to bind        to B and that is suitably labelled with a fluorophore or        chromophore group or other chemical moiety, such as biotin for        easy detection in an ELISA or FACS (Fluorescent activated cell        sorting) or other format (the fluorophore for fluorescence        detection, the chromophore for light absorption detection, the        biotin for streptavidin-mediated ELISA detection). Typically,        the reference molecule C is kept at a fixed concentration and        the concentration of A is varied for a given concentration or        amount of B. As a result an IC₅₀ value is obtained corresponding        to the concentration of A at which the signal measured for C in        absence of A is halved. Provided K_(D ref), the K_(D) of the        reference molecule, is known, as well as the total concentration        c_(ref) of the reference molecule, the apparent K_(D) for the        interaction A-B can be obtained from following formula:        K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if        c_(ref)<<K_(D ref), K_(D)IC₅₀. Provided the measurement of the        IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)        fixed) for the binders that are compared, the strength or        stability of a molecular interaction can be assessed by the IC₅₀        and this measurement is judged as equivalent to K_(D) or to        apparent K_(D) throughout this text.-   p) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020079. As also mentioned in paragraph o) on page 57 of WO    08/020079, the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC).    Reference is for example made to the Experimental Part below, as    well as to the standard handbooks, such as Kenneth, A et al:    Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists    and Peters et al, Pharmacokinete analysis: A Practical Approach    (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D    Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The    terms “increase in half-life” or “increased half-life” as also as    defined in paragraph o) on page 57 of WO 08/020079 and in particular    refer to an increase in the t1/2-beta, either with or without an    increase in the t1/2-alpha and/or the AUC or both.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An immunoglobulin single variable domain or polypeptide is said    to be “specific for” a first target or antigen compared to a second    target or antigen when it binds to the first antigen with an    affinity/avidity (as described above, and suitably expressed as a    K_(D) value, K_(A) value, K_(off) rate and/or K_(on) rate) that is    at least 10 times, such as at least 100 times, and preferably at    least 1000 times, and up to 10000 times or more better than the    affinity with which said amino acid sequence or polypeptide binds to    the second target or polypeptide. For example, the first antigen may    bind to the target or antigen with a K_(D) value that is at least 10    times less, such as at least 100 times less, and preferably at least    1000 times less, such as 10000 times less or even less than that,    than the K_(D) with which said amino acid sequence or polypeptide    binds to the second target or polypeptide. Preferably, when an    immunoglobulin single variable domain or polypeptide is “specific    for” a first target or antigen compared to a second target or    antigen, it is directed against (as defined herein) said first    target or antigen, but not directed against said second target or    antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an immunoglobulin    single variable domain or polypeptide to interfere with the binding    of the natural ligand to its receptor(s). The extent to which an    immunoglobulin single variable domain or polypeptide of the    invention is able to interfere with the binding of another compound    such as the natural ligand to its target, e.g., CXCR4, and therefore    whether it can be said to cross-block according to the invention,    can be determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a FACS- or an    ELISA-based approach or ALPHASCREEN® to measure competition between    the labelled (e.g., His tagged or biotinylated) immunoglobulin    single variable domain or polypeptide according to the invention and    the other binding agent in terms of their binding to the target. The    experimental part generally describes suitable FACS-, ELISA- or    ALPHASCREEN®-displacement-based assays for determining whether a    binding molecule cross-blocks or is capable of cross-blocking an    immunoglobulin single variable domain or polypeptide according to    the invention. It will be appreciated that the assay can be used    with any of the immunoglobulin single variable domains or other    binding agents described herein. Thus, in general, a cross-blocking    amino acid sequence or other binding agent according to the    invention is for example one which will bind to the target in the    above cross-blocking assay such that, during the assay and in the    presence of a second amino acid sequence or other binding agent of    the invention, the recorded displacement of the immunoglobulin    single variable domain or polypeptide according to the invention is    between 60% and 100% (e.g., in ELISA/ALPHASCREEN® based competition    assay) or between 80% to 100% (e.g., in FACS based competition    assay) of the maximum theoretical displacement (e.g. displacement by    cold (e.g., unlabeled) immunoglobulin single variable domain or    polypeptide that needs to be cross-blocked) by the to be tested    potentially cross-blocking agent that is present in an amount of    0.01 mM or less (cross-blocking agent may be another conventional    monoclonal antibody such as IgG, classic monovalent antibody    fragments (Fab, scFv)) and engineered variants (e.g., diabodies,    triabodies, minibodies, VHHs, dAbs, VHs, VLs).-   t) An amino acid sequence such as e.g. an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be a “VHH1 type immunoglobulin single variable domain” or “VHH type    1 sequence”, if said VHH1 type immunoglobulin single variable domain    or VHH type 1 sequence has 85% identity (using the VHH1 consensus    sequence as the query sequence and use the blast algorithm with    standard setting, i.e., blosom62 scoring matrix) to the VHH1    consensus sequence    (QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSS-DGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA),    and mandatorily has a cysteine in position 50, i.e., C50 (using    Kabat numbering).-   u) An amino acid sequence such as e.g., an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be “cross-reactive” for two different antigens or antigenic    determinants (such as serum albumin from two different species of    mammal, such as human serum albumin and cynomolgus monkey serum    albumin) if it is specific for (as defined herein) both these    different antigens or antigenic determinants.-   v) As further described in paragraph q) on pages 58 and 59 of WO    08/020079 (incorporated herein by reference), the amino acid    residues of an immunoglobulin single variable domain are numbered    according to the general numbering for V_(H) domains given by Kabat    et al. (“Sequence of proteins of immunological interest”, US Public    Health Services, NIH Bethesda, Md., Publication No. 91), as applied    to V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication), and accordingly FR1 of    an immunoglobulin single variable domain comprises the amino acid    residues at positions 1-30, CDR1 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    31-35, FR2 of an immunoglobulin single variable domain comprises the    amino acids at positions 36-49, CDR2 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    50-65, FR3 of an immunoglobulin single variable domain comprises the    amino acid residues at positions 66-94, CDR3 of an immunoglobulin    single variable domain comprises the amino acid residues at    positions 95-102, and FR4 of an immunoglobulin single variable    domain comprises the amino acid residues at positions 103-113.-   w) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.-   x) The half maximal inhibitory concentration (IC₅₀) is a measure of    the effectiveness of a compound in inhibiting a biological or    biochemical function, e.g. a pharmacological effect. This    quantitative measure indicates how much of the ISV or Nanobody    (inhibitor) is needed to inhibit a given biological process (or    component of a process, i.e. an enzyme, cell, cell receptor,    chemotaxis, HIV entry, HIV replication, HIV reverse transcriptase    activity, etc.) by half. In other words, it is the half maximal    (50%) inhibitory concentration (IC) of a substance (50% IC, or    IC₅₀). The IC₅₀ of a drug can be determined by constructing a    dose-response curve and examining the effect of different    concentrations of antagonist such as the ISV or Nanobody of the    invention on reversing agonist activity. IC₅₀ values can be    calculated for a given antagonist such as the ISV or Nanobody of the    invention by determining the concentration needed to inhibit half of    the maximum biological response of the agonist.    -   The term half maximal effective concentration (EC₅₀) refers to        the concentration of a compound which induces a response halfway        between the baseline and maximum after a specified exposure        time. In the present context it is used as a measure of a        polypeptide's, ISV's or Nanobody's potency. The EC₅₀ of a graded        dose response curve represents the concentration of a compound        where 50% of its maximal effect is observed. Concentration is        preferably expressed in molar units.    -   In biological systems, small changes in ligand concentration        typically result in rapid changes in response, following a        sigmoidal function. The inflection point at which the increase        in response with increasing ligand concentration begins to slow        is the EC₅₀. This can be determined mathematically by derivation        of the best-fit line. Relying on a graph for estimation is        convenient in most cases. In case the EC₅₀ is provided in the        experimental section, the experiments were designed to reflect        the KD as accurate as possible. In other words, the EC₅₀ values        may then be considered as KD values. The term “average KD”        relates to the average KD value obtained in at least 1, but        preferably more than 1, such as at least 2 experiments. The term        “average” refers to the mathematical term “average” (sums of        data divided by the number of items in the data).    -   It is also related to IC₅₀ which is a measure of a compound's        inhibition (50% inhibition). For competition binding assays and        functional antagonist assays IC₅₀ is the most common summary        measure of the dose-response curve. For agonist/stimulator        assays the most common summary measure is the EC₅₀.

The synergistic inhibition of HIV infection by different classes ofsmall molecule inhibitors is known. In general these inhibitors aredirected against virus derived components, but less to the cellular hostcomponents involved in HIV infection, such as the human receptor (CD4)and co-receptor(s). The present inventors demonstrated that binding by abispecific polypeptide directed against a co-receptor (CR) and thereceptor CD4 resulted in a synergy of the two binding moieties ininhibiting HIV infection (see Example 4). Surprisingly, the bispecificpolypeptides were exceptionally more effective than a combination of thetwo individual moieties (see Example 8).

The present invention relates to particular polypeptides, also referredto as “polypeptides of the invention”, “bispecific polypeptides”,“bispecific constructs” or “bispecific Nanobody constructs” thatcomprise or essentially consist of (i) a first building block consistingessentially of a first immunoglobulin single variable domain, whereinsaid first immunoglobulin single variable domain binds a first target,an HIV receptor, such as CD4, on the surface of a cell; and (ii) asecond building block consisting essentially of a second immunoglobulinsingle variable domain, wherein said second immunoglobulin singlevariable domain binds a second target, preferably an HIV co-receptor, onthe surface of a cell, and wherein said CR is not CD4.

“Synergy” between two or more agents refers to the combined effect ofthe agents which is greater than their additive effects. Illustratively,agents may be peptides, proteins, such as antibodies, small molecules,organic compounds, and drug forms thereof. Synergistic, additive orantagonistic effects between agents may be quantified by analysis of thedose-response curves using the Combination Index (CI) method. A CI valuegreater than 1 indicates antagonism; a CI value equal to 1 indicates anadditive effect; and a CI value less than 1 indicates a synergisticeffect. In one embodiment, the CI value of a synergistic interaction isless than 0.9. In another embodiment, the CI value is less than 0.8. Ina preferred embodiment, the CI value is less than 0.7 (cf. Example 4 andChou and Talalay, 1984, which is incorporated herein by reference). Theterm antagonist is well known in the art. In essence, the termantagonist relates to a substance that acts against and blocks anaction. For instance, antagonists have affinity but no efficacy fortheir cognate receptors, and binding will disrupt the interaction andinhibit the function of an agonist or inverse agonist at receptors.

“HIV” refers to the human immunodeficiency virus. HIV shall include,without limitation, HIV-1 and HIV-2. HIV-1 includes but is not limitedto extracellular virus particles and the forms of HIV-1 associated withHIV-1 infected cells. The human immunodeficiency virus (HIV) may beeither of the two known types of HIV (HIV-1 or HIV-2). The HIV-1 virusmay represent any of the known major subtypes (classes A, B, C, D, E, F,G, H, or J), outlying subtype (Group 0), or an as yet to be determinedsubtype of HIV-1. HIV-1 JRFL is a strain that was originally isolated atautopsy from the brain tissue of an AIDS patient. The virus has beencloned and the DNA sequences of its envelope glycoproteins are known(GenBank Accession No. U63632). In terms of sensitivity to inhibitors ofviral entry, HIV-1 JFRL is known to be highly representative of primaryHIV-1 isolates. “JRCSF” refers to a HIV-1 isolate of subtype B. JRCSF isa strain originally isolated from cerebral spinal fluid and brain tissueof an AIDS patient (Science 236, 819-822, 1987). The virus has beencloned and its genome DNA sequence is known (GenBank Accession No.M38429). Unlike HIV isolate JRFL, JRCSF does not productively infectmacrophages. The CXCR4-using (X4) HIV-1 clone NL4.3 was obtained fromthe National Institutes of Health NIAID AIDS Reagent program (Bethesda,Md.). The CCR5-using (R5) HIV-1 strain BaL was obtained from the MedicalResearch Council AIDS reagent project (Herts, UK). The dual-tropic(R5/X4) HIV-1 HE strain was initially isolated from a patient at theUniversity Hospital in Leuven.

The polypeptides of the invention are designed to inhibit HIV infection.

The term “HIV infection” refers to the entry of HIV into a susceptiblecell. Infection of cells by human immunodeficiency virus type 1 (HIV-1)is mediated by the viral envelope (Env) glycoproteins gp120 and gp41,which are expressed as a non-covalent, oligomeric complex on the surfaceof virus and virally infected cells. Entry of the virus into targetcells proceeds through a cascade of events at the cell surface thatinclude (1) high-affinity interaction between the HIV surfaceglycoprotein gp120 to the cell surface receptor CD4, (2) Env binding tofusion co-receptors, and (3) conformational changes in the viraltransmembrane glycoprotein gp41, which mediates fusion of the viral andcellular membranes.

In essence, inhibiting HIV infection relates to inhibiting at least onefunction in the HIV life cycle, preferably more than one function. Thesefunctions include, for instance, binding of HIV to the receptor CD4,binding of HIV to the co-receptor, entry of HIV into a target cell,replication of HIV, HIV reverse transcriptase activity, HIV-induced celldeath, and/or HIV-induced cell-cell syncytia formation. Preferably, thetransmission of HIV is inhibited. Inhibition of HIV infection can bemeasured by various methods, both in vitro and in vivo. Preferably,inhibiting HIV infection results in reducing the viral load or themaintenance of a reduced viral load and preferably by ameliorating themedical condition of the HIV infected subject. The term “viral load”refers to the amount of HIV particles in a sample of blood, generallyindicated as the number of copies per ml blood. For instance, a viralload of more than 100,000 copies/ml would be considered high, while aviral load of less than 10,000 copies/ml would be considered low.Preferably, the viral load is reduced to undetectable levels (<50 copiesper ml).

Inhibition, as used herein, includes both complete and partialinhibition. Thus, the disclosure embraces polypeptides comprising two ormore immunoglobulin single variable domains that inhibit binding of HIVto CD4 and/or a co-receptor, such as e.g. CXCR4, by more than 1%, morethan 2%, more than 5%, more than 10%, more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90%, or up to 100% inhibition.

The present invention thus relates to a polypeptide as described herein,wherein said polypeptide inhibits HIV infection by about 10%, 20%, 30%,40%, 50%, 60%, 80%, 90% and preferably 95% or more, such as 100% (asmeasured in a HIV infection assay).

It should be appreciated that inhibition can also be expressed in IC₅₀(Inhibitory Concentration), defined as the inhibitory concentration atwhich 50% of HIV is inhibited from binding to a receptor or co-receptor,such as e.g. CXCR4. In some embodiments, the IC₅₀ of inhibition of HIVbinding to CD4 or a co-receptor such as e.g. CXCR4 by a polypeptidecomprising two or more immunoglobulin single variable domains is lowerthan 500 μM, lower than 100 μM, lower than 50 μM, lower than 10 μM,lower than 50 μM, lower than 1 μM, lower than 500 nM, lower than 100 nM,lower than 90 nM, lower than 80 nM, lower than 70 nM, lower than 60 nM,lower than 50 nM, lower than 40 nM, lower than 30 nM, lower than 20 nM,lower than 10 nM, lower than 9 nM, lower than 8 nM, lower than 7 nM,lower than 6 nM, lower than 5 nM, lower than 4 nM, lower than 3 nM,lower than 2 nM, lower than 1 nM, lower than 100 pM, lower than 50 pM,lower than 10 pM, lower than 5 pM, 4, 3, 2, 1, 0.5 pM, or even less suchas less than 0.4 pM.

In some embodiments, the IC₅₀ of inhibition of HIV binding to CD4 and/ora co-receptor such as e.g. CXCR4 by the polypeptide of the invention islower than 50 nM. In some embodiments, the IC₅₀ of inhibition of HIVbinding to CD4 and/or a co-receptor such as e.g. CXCR4 by thepolypeptide of the invention is lower than 1 nM. In some embodiments,the IC₅₀ of inhibition of HIV binding to CD4 and/or a co-receptor suchas e.g. CXCR4 by the polypeptide of the invention is lower than 10 pM.

Similarly, inhibition can also be expressed by EC₅₀ (cf. supra).Accordingly, the present invention relates to a polypeptide as describedherein, wherein the average EC₅₀ value of HIV inhibition is of between10 nM and 0.1 pM, such as at an average EC₅₀ value of 10 nM or less,even more preferably at an average EC₅₀ value of 9 nM or less, such asless than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as less than400, 300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5pM, or even less such as less than 0.4 pM.

In one aspect, the disclosure provides polypeptides comprising two ormore immunoglobulin single variable domains that inhibit binding of HIVto CD4 and/or a co-receptor such as e.g. CXCR4.

Accordingly, the present invention relates to polypeptides as describedherein, wherein said polypeptides inhibit HIV fusion with CD4 CXCR4⁺cells.

The efficacy of an anti-HIV compound in inhibiting HIV infection can bemeasured by various methods, ubiquitously known to the person skilled inthe art. For instance, cell-based anti-viral assays can be performedwith either transformed T-cell lines (SupT1, H9, Molt4), primaryblood-derived mononuclear cells (PBMCs) or macrophages. Experimentalreadout of HIV replication can be performed by ELISA of p24 viralantigen, monitoring reverse transcriptase, tat-expression, monitoringreporter virus or by intracellular or surface staining of viral antigens(e.g. Gag protein or Env, respectively). Single-cycle infectivity assaysusing a variety of stable reporter cell lines expressing HIV receptorand co-receptors can be used to evaluate infectivity of HIV or HIV-basedpseudotypes using single cycle or quasi-single cycle assays. Theefficacy of an anti-HIV compound in inhibiting HIV infection can also bemeasured by in vitro models for HIV including assays evaluating theability to reactivate viral replication from latently infected cells andproduce HIV virus; assays in synchronized infections, evaluation ofviral entry inhibition, determination of HIV reverse transcriptaseactivity, integrase assays; characterization of resistant variantsincluding determination of genotypic and phenotypic variations;evaluation of HIV protease activity, entry assays, integration assays,antibody neutralization assays, co-receptor determination, HIV-induceddown-modulation of CD4 and class I MHC, anti-HIV activity in chronicallyinfected cells (with established HIV replication), evaluation of CPEeffect in infected cells (syncytia formation, apoptosis, directkilling). All these methods are established methods and well known tothe person skilled in the art.

In some embodiments, the (inhibition of) binding of HIV to CD4 or a CRsuch as e.g. CXCR4 is determined by biochemical assays. In someembodiments, the (inhibition of) binding of HIV to CD4 or a CR such ase.g. CXCR4 is determined by functional assays. In some embodiments, theassays are competition assays, e.g., with a natural ligand, and/or mayinclude comparisons to a standard.

In some embodiments, biochemical assays include a step of contacting apolypeptide encompassing the complete or partial CD4 or CR sequence,such as e.g. a CXCR4 sequence, or cells expressing such sequences, withHIV or one of more HIV proteins, or protein fragments of HIV, that canbind to CD4 or a CR such as e.g. CXCR4. Binding can subsequently bedetermined through a variety of methods including ELISA, e.g., by usingantibodies that detect the presence of one or both binding partners,surface plasmon resonance, or fluorescence based techniques such asFRET.

Functional assays include assays based on the suppression or increase ofone or more biological functions of CD4 and/or a CR, such as e.g. CXCR4,and/or HIV, and are generally performed on live cells (e.g., cellsexpressing CD4 and/or CXCR4, cf. experimental section). For instance,CXCR4 activation, e.g., by natural ligand binding, triggers cellsignaling pathways that are suppressed when CXCR4 is bound by HIV. Thus,monitoring the downstream events of such pathways e.g., the level ofcAMP, provides a functional assay that allows for the determination ofbinding of CXCR4 by HIV and/or the displacement of natural ligand (SeeExample section). Alternatively, binding of a cell expressing CD4 and/ora CR, such as e.g. CXCR4, by HIV may result in a change in cellularfunction (e.g., phagocytosis) and inhibition of HIV binding can bemonitored by quantifying the cellular function induced by HIV-binding.

During HIV transmission, CD4⁺ T-cells can not only become infected bycell-free virions but, importantly, also by close cell-cell contactswith donor HIV-infected T-cells. As set out in Example 9, this can bemeasured based on the appearance of giant cells or syncytia in the cellco-cultures.

Accordingly, the present invention relates to polypeptides as describedherein, wherein said polypeptides inhibit HIV-induced cell-cell syncytiaformation.

The polypeptides of the present invention provide a more specificinhibition of HIV infection than prior art antibodies. Preferably, thebispecific polypeptides of the invention comprise at least two bindingmoieties, such as for instance two building blocks, ISVs or Nanobodies,wherein at least the first binding moiety (functional ISV) is specificfor CD4.

The terms polypeptide of the invention, bispecific polypeptide,bispecific construct, bispecific Nanobody construct, bispecific andbispecific antibody are used interchangeably herein.

Accordingly, the present invention relates to a polypeptide comprising afirst and a second immunoglobulin single variable domain (ISV), wherein

-   -   said first ISV binds to CD4 present on the surface of a cell;    -   said second ISV binds to a co-receptor (CR) present on the        surface of said cell; and wherein said CR is not CD4.

In one aspect, the disclosure provides polypeptides that include one ormore immunoglobulin single variable domains that inhibit binding of HIVto CXCR4.

In some embodiments, the polypeptides comprise at least two or moreimmunoglobulin single variable domains disclosed herein. In someembodiments, the polypeptides essentially consist of two or moreimmunoglobulin single variable domains disclosed herein. A polypeptidethat “essentially consists of” two or more immunoglobulin singlevariable domains, is a polypeptide that in addition to the two or moreimmunoglobulin single variable domains disclosed herein does not haveadditional immunoglobulin single variable domains. For instance, apolypeptide that essentially consists of two immunoglobulin singlevariable domains does not include any additional immunoglobulin singlevariable domains. However, it should be appreciated that a polypeptidethat essentially consists of two or more immunoglobulin single variabledomains may include additional functionalities, such as a label, atoxin, one or more linkers, a binding sequence, etc. These additionalfunctionalities include both amino acid based and non-amino acid basedgroups. In some embodiments, the polypeptides consist of one or moreimmunoglobulin single variable domains disclosed herein. It should beappreciated that the terms “polypeptide construct” and “polypeptide” canbe used interchangeably herein (unless the context clearly dictatesotherwise).

In some embodiments, the polypeptides include multivalent ormultispecific constructs comprising immunoglobulin single variabledomains disclosed herein. In some embodiments, the polypeptides compriseone or more antibody based-scaffolds and/or non-antibody based scaffoldsdisclosed herein. In some embodiments, the polypeptides comprise a serumbinding protein moiety. In some embodiments, the serum binding proteinmoiety is an immunoglobulin single variable domain. In some embodiments,the immunoglobulin single variable domain is a Nanobody®, a V_(HH), ahumanized V_(HH) or a camelized V_(H).

Two or more immunoglobulin single variable domains can be combined in asingle polypeptide, resulting in a multivalent and/or multispecificpolypeptide, e.g. a bispecific polypeptide. Multivalent and/ormultispecific polypeptides allow for improved avidity of the construct(i.e., for a desired antigen) as compared to a single immunoglobulinsingle variable domains, and/or for constructs that can bind to two ormore different antigens. In some embodiments, the multispecificpolypeptides include two or more immunoglobulin single variable domainsthat bind to the same target, thereby increasing the affinity forbinding to a single antigen. In some embodiments, the polypeptide isbiparatopic. The bispecific or multispecific polypeptides of the presentinvention comprise or essentially consist of at least two buildingblocks, e.g. ISVs, of which the first building block, e.g. the firstISV, has an increased affinity for its antigen, i.e. the first target,upon binding by the second building block, e.g. the second ISV, to itsantigen, i.e. the second target. Such increased affinity (apparentaffinity), due to avidity effects, is also called ‘conditionalbispecific or multispecific binding’. Such bispecific or multispecificpolypeptide is also called ‘a conditionally binding bispecific ormultispecific polypeptide of the invention’.

It will be appreciated that the order of the first building block andthe second building block on the polypeptide (orientation) can be chosenaccording to the needs of the person skilled in the art, as well as therelative affinities which may depend on the location of these buildingblocks in the polypeptide. Whether the polypeptide comprises a linker,is a matter of design choice. However, some orientations, with orwithout linkers, may provide preferred binding characteristics incomparison to other orientations. For instance, the order of the firstand the second building block in the polypeptide of the invention can be(from N-terminus to C-terminus): (i) first building block (e.g. a firstISV such as a first Nanobody)—[linker]—second building block (e.g. asecond ISV such as a second Nanobody); or (ii) second building block(e.g. a second ISV such as a second Nanobody)-[linker]-first buildingblock (e.g. a first ISV such as a first Nanobody); (wherein the linkeris optional). All orientations are encompassed by the invention.Polypeptides that contain an orientation of building blocks thatprovides desired (binding) characteristics can be easily identified byroutine screening, for instance as exemplified in the experimentalsection.

As noted before, the inventors demonstrated inter alia thataccomplishing resistance by HIV against the bispecific polypeptide isextremely difficult, even in a forced laboratory setting (see Example8). It was surprisingly observed that even in a HIV strain maderesistant against one target, e.g. the anti-CD4 moiety, the bispecificpolypeptide was still potent in inhibiting HIV infection. Hence, thisproperty expands the use of a bispecific polypeptide to a possibleefficacy against heterogeneous strains not inherently resistant to onemoiety agent and another HIV strain not inherently resistant againstanother moiety.

The invention relates to a method of inhibiting HIV infection of asusceptible cell in a subject by an HIV virus that is resistant, or hasbecome resistant, to a CD4 antagonist, which comprises subjecting thesusceptible cell to an effective HIV infection inhibiting dose of apolypeptide as described herein (which inhibits HIV fusion with CD4CXCR4⁺ cells), preferably wherein the effective HIV infection inhibitingdose comprises from 0.1 mg per kg to 25 mg per kg of the subject's bodyweight, so as to thereby inhibit the infection of the susceptible cellby HIV1 that is resistant, or has become resistant, to the CD4antagonist.

As used herein, the term “potency” is a measure of an agent, such as apolypeptide, ISV or Nanobody, its biological activity. Potency of anagent can be determined by any suitable method known in the art, such asfor instance as described in the experimental section. Cell culturebased potency assays are often the preferred format for determiningbiological activity since they measure the physiological responseelicited by the agent and can generate results within a relatively shortperiod of time. Various types of cell based assays, based on themechanism of action of the product, can be used, including but notlimited to proliferation assays, cytotoxicity assays, reporter geneassays, cell surface receptor binding assays and assays to measureinduction/inhibition of functionally essential protein or other signalmolecule (such as phosphorylated proteins, enzymes, cytokines, cAMP andthe like), all well known in the art. Results from cell based potencyassays can be expressed as “relative potency” as determined bycomparison of the bispecific polypeptide of the invention to theresponse obtained for the corresponding reference monovalent ISV, e.g. apolypeptide comprising only one ISV or one Nanobody, optionally furthercomprising an irrelevant Nanobody (cf. experimental section).

A compound, e.g. the bispecific polypeptide, is said to be more potentthan the reference compound, e.g. a compound such as a small molecule ora (conventional) antibody directed at the same target or thecorresponding monovalent or monospecific ISV or Nanobody or polypeptidecomprising the corresponding monovalent or monospecific ISV or Nanobody,when the response obtained for the compound, e.g. the bispecificpolypeptide, is at least 2 times, but preferably at least 3 times, suchas at least 4 times, at least 5 times, at least 6 times, at least 7times, at least 8 times, at least 9 times, at least 10 times, at least15 times, at least 20 times, at least 25 times, at least 50 times, atleast 75 times, at least 100 times, and even more preferably even atleast 200 times, or even at least 500 times, or even 1000 times better(e.g. functionally better) than the response by the reference compound,e.g. the corresponding monovalent ISV or Nanobody in a given assay.

The cell of the invention relates in particular to mammalian cells, andpreferably to primate cells and even more preferably to human cells.

The cell is preferably an immune cell, such as a T-helper cell,monocyte, macrophage, or dendritic cell, preferably a CD4⁺ T-helper cell(also known as CD4 cell, CD4⁺ cell, T-helper cell or T4 cell),preferably a CD4⁺ CXCR4⁺ cell, even more preferably a human cell. Insome embodiments, the cell is in vivo. In some embodiments, the cell isin vitro.

The membrane (also called plasma membrane or phospholipid bilayer)surrounds the cytoplasm of a cell, which is the outer boundary of thecell, i.e. the membrane is the surface of the cell. This membrane servesto separate and protect a cell from its surrounding environment and ismade mostly from a double layer of phospholipids. Embedded within thismembrane is a variety of protein molecules, such as channels, pumps andcellular receptors. Since the membrane is fluid, the protein moleculescan travel within the membrane.

For a general description of immunoglobulin single variable domains,reference is made to the further description below, as well as to theprior art cited herein. In this respect, it should however be noted thatthis description and the prior art mainly describes immunoglobulinsingle variable domains of the so-called “V_(H)3 class” (i.e.,immunoglobulin single variable domains with a high degree of sequencehomology to human germline sequences of the V_(H)3 class such as DP-47,DP-51 or DP-29), which form a preferred aspect of this invention. Itshould, however, be noted that the invention in its broadest sensegenerally covers any type of immunoglobulin single variable domains andfor example also covers the immunoglobulin single variable domainsbelonging to the so-called “V_(H)4 class” (i.e., immunoglobulin singlevariable domains with a high degree of sequence homology to humangermline sequences of the V_(H)4 class such as DP-78), as for exampledescribed in WO 07/118670.

Generally, immunoglobulin single variable domains (in particular V_(HH)sequences and sequence optimized immunoglobulin single variable domains)can in particular be characterized by the presence of one or more“Hallmark residues” (as described herein) in one or more of theframework sequences (again as further described herein).

Thus, generally, an immunoglobulin single variable domain can be definedas an amino acid sequence with the (general) structure (cf. formula 1below)

FR1-CDR1-FR2-CR2CDR2-FR3-CDR-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively.

In a preferred aspect, the invention provides polypeptides comprising atleast an immunoglobulin single variable domain that is an amino acidsequence with the (general) structure

FR1-CDR1-FR2-CR2CDR2-FR3-CDR-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) at least one of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-1 below;    and in which:-   ii) said amino acid sequence has at least 80%, more preferably 90%,    even more preferably 95% amino acid identity with at least one of    the immunoglobulin single variable domains as shown in WO    2009/138519 (see SEQ ID NOs: 1 to 125 in WO 2009/138519), in which    for the purposes of determining the degree of amino acid identity,    the amino acid residues that form the CDR sequences (indicated with    X in the sequences) are disregarded; and in which:-   iii) the CDR sequences are generally as further defined herein    (e.g., the CDR1, CDR2 and CDR3 in a combination as can be determined    with the information provided herein, noting that the CDR    definitions are calculated according to the Kabat numbering system).

TABLE A-1 Hallmark Residues in VHHs Position Human V_(H)3 HallmarkResidues  11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, predominantlyL Y, N, P, I; preferably L  37 V, I, F; F⁽¹⁾, Y, V, L, A, H, S, I, W, C,N, G, usually V D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾, Q⁽³⁾, G⁽²⁾,D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾, E⁽³⁾ or Q⁽³⁾;most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F, G, Q, S, E,T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y F⁽¹⁾, L⁽¹⁾ or W⁽²⁾G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W⁽²⁾, L⁽¹⁾or F⁽¹⁾  83 R or K; R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, G, M, usually R L,A, D, Y, H; preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, S,H, L, A, V, I, T, F, D, R, Y, predominantly A N, Q, G, E; preferably P103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q, P⁽⁶⁾, E, C;preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108 L, Mor T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H; predominantly L preferably Qor L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, in combinationwith KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW at positions44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. as KEREL,KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQRE atpositions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW.

The immunoglobulins of the invention may also contain a C-terminalextension (X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2,3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an(preferably naturally occurring) amino acid residue that isindependently chosen, and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I)), for which reference is made to WO 12/175741 and USprovisional applications, all entitled “Improved immunoglobulin variabledomains”: U.S. 61/994,552 filed May 16, 2014; U.S. 61/014,015 filed Jun.18, 2014; U.S. 62/040,167 filed Aug. 21, 2014; and U.S. 62/047,560,filed Sep. 8, 2014 (all assigned to Ablynx N.V.).

Again, such immunoglobulin single variable domains may be derived in anysuitable manner and from any suitable source, and may for example benaturally occurring V_(HH) sequences (i.e., from a suitable species ofCamelid, e.g., llama) or synthetic or semi-synthetic VHs or VLs (e.g.,from human). Such immunoglobulin single variable domains may include“humanized” or otherwise “sequence optimized” VHHs, “camelized”immunoglobulin sequences (and in particular camelized heavy chainvariable domain sequences, i.e., camelized VHs), as well as human VHs,human VLs, camelid VHHs that have been altered by techniques such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, veneering,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the foregoing as further describedherein. As mentioned herein, a particularly preferred class ofimmunoglobulin single variable domains of the invention comprisesimmunoglobulin single variable domains with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above). This can beperformed in a manner known per se, which will be clear to the skilledperson, for example on the basis of the further description herein andthe prior art on humanization referred to herein. Again, it should benoted that such humanized immunoglobulin single variable domains of theinvention can be obtained in any suitable manner known per se and thusare not strictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(HH) domain as astarting material.

Another particularly preferred class of immunoglobulin single variabledomains of the invention comprises immunoglobulin single variabledomains with an amino acid sequence that corresponds to the amino acidsequence of a naturally occurring V_(H) domain, but that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe description herein. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theV_(H)—V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see also for example WO 94/04678 and Daviesand Riechmann (1994 and 1996)). Preferably, the V_(H) sequence that isused as a starting material or starting point for generating ordesigning the camelized immunoglobulin single variable domains ispreferably a V_(H) sequence from a mammal, more preferably the V_(H)sequence of a human being, such as a V_(H)3 sequence. However, it shouldbe noted that such camelized immunoglobulin single variable domains ofthe invention can be obtained in any suitable manner known per se andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring V_(H) domain asa starting material.

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” immunoglobulin singlevariable domain of the invention, respectively. This nucleic acid canthen be expressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention. Alternatively,based on the amino acid sequence of a naturally occurring V_(HH) domainor V_(H) domain, respectively, the amino acid sequence of the desiredhumanized or camelized immunoglobulin single variable domains of theinvention, respectively, can be designed and then synthesized de novousing techniques for peptide synthesis known per se. Also, based on theamino acid sequence or nucleotide sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized immunoglobulin singlevariable domains of the invention, respectively, can be designed andthen synthesized de novo using techniques for nucleic acid synthesisknown per se, after which the nucleic acid thus obtained can beexpressed in a manner known per se, so as to provide the desiredimmunoglobulin single variable domains of the invention.

Generally, proteins or polypeptides that comprise or essentially consistof a single building block, single immunoglobulin single variable domainor single Nanobody will be referred to herein as “monovalent” proteinsor polypeptides or as “monovalent constructs”, or as monovalent buildingblock, monovalent immunoglobulin single variable domain or monovalentNanobody, respectively. Proteins and polypeptides that comprise oressentially consist of two or more immunoglobulin single variabledomains (such as at least two immunoglobulin single variable domains ofthe invention) will be referred to herein as “multivalent” proteins orpolypeptides or as “multivalent constructs”, and these provide certainadvantages compared to the corresponding monovalent immunoglobulinsingle variable domains of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein. The polypeptides of the invention are “multivalent”,i.e. comprising two or more building blocks or ISVs of which at leastthe first building block, ISV or Nanobody and the second building block,ISV or Nanobody are different, and directed against different targets,such as antigens or antigenic determinants. Polypeptides of theinvention that contain at least two building blocks, ISVs or Nanobodies,in which at least one building block, ISV or Nanobody is directedagainst a first antigen (i.e., against the first target, such as e.g.CD4) and at least one building block, ISV or Nanobody is directedagainst a second antigen (i.e., against the second target which isdifferent from the first target, such as e.g. a CR, e.g. CXCR4), willalso be referred to as “multispecific” polypeptides of the invention,and the building blocks, ISVs or Nanobodies present in such polypeptideswill also be referred to herein as being in a “multivalent format”.Thus, for example, a “bispecific” polypeptide of the invention is apolypeptide that comprises at least one building block, ISV or Nanobodydirected against a first target (e.g. CD4) and at least one furtherbuilding block, ISV or Nanobody directed against a second target (i.e.,directed against a second target different from said first target, suchas e.g. CR, e.g. CXCR4), whereas a “trispecific” polypeptide of theinvention is a polypeptide that comprises at least one building block,ISV or Nanobody directed against a first target (e.g., CD4), a secondbuilding block, ISV or Nanobody directed against a second targetdifferent from said first target (e.g. CR, such as e.g. CXCR4) and atleast one further building block, ISV or Nanobody directed against athird antigen (i.e., different from both the first and the secondtarget), such as, for instance, serum albumin; etc. As will be clearfrom the description, the invention is not limited to bispecificpolypeptides, in the sense that a multispecific polypeptide of theinvention may comprise at least a first building block, ISV or Nanobodyagainst a first target, a second building block, ISV or Nanobody againsta second target and any number of building blocks, ISVs or Nanobodiesdirected against one or more targets, which may be the same or differentfrom the first and/or second target, respectively. The building blocks,ISVs or Nanobodies can optionally be linked via linker sequences.

Accordingly, the present invention also relates to a trispecific ormultispecific polypeptide, comprising or essentially consisting of atleast three binding moieties, such as three ISVs, wherein at least oneof said at least three binding moieties is directed against a firsttarget with a low, moderate of high affinity, at least one of said atleast three binding moieties is directed against a second target with ahigh affinity and at least a third binding moiety increasing half-life,such as e.g. an albumin binder.

As will be clear from the further description above and herein, theimmunoglobulin single variable domains of the invention can be used as“building blocks” to form polypeptides of the invention, e.g., bysuitably combining them with other groups, residues, moieties or bindingunits, in order to form compounds or constructs as described herein(such as, without limitations, the bi-/tri-/tetra-/multivalent andbi-/tri-/tetra-/multispecific polypeptides of the invention describedherein) which combine within one molecule one or more desired propertiesor biological functions.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more immunoglobulin single variable domains of the invention tothe one or more further groups, residues, moieties or binding units,optionally via the one or more suitable linkers, so as to provide thecompound or polypeptide of the invention. Polypeptides of the inventioncan also be prepared by a method which generally comprises at least thesteps of providing a nucleic acid that encodes a polypeptide of theinvention, expressing said nucleic acid in a suitable manner, andrecovering the expressed polypeptide of the invention. Such methods canbe performed in a manner known per se, which will be clear to theskilled person, for example on the basis of the methods and techniquesfurther described herein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedimmunoglobulin single variable domains form a further aspect of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional immunoglobulin single variable domains,such that the compound or construct is a (fusion) protein or (fusion)polypeptide. In a preferred but non-limiting aspect, said one or moreother groups, residues, moieties or binding units are immunoglobulinsequences. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, immunoglobulin single variable domains that aresuitable for use as a domain antibody, single domain antibodies,immunoglobulin single variable domains (ISVs) that are suitable for useas a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies.Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreimmunoglobulin single variable domains of the invention so as to providea “derivative” of an amino acid sequence or polypeptide of theinvention, as further described herein.

Also within the scope of the present invention are compounds orconstructs, which comprise or essentially consist of one or morederivatives as described herein, and optionally further comprise one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are immunoglobulin singlevariable domains. In the compounds or constructs described above, theone or more immunoglobulin single variable domains of the invention andthe one or more groups, residues, moieties or binding units may belinked directly to each other and/or via one or more suitable linkers orspacers. For example, when the one or more groups, residues, moieties orbinding units are immunoglobulin single variable domains, the linkersmay also be immunoglobulin single variable domains, so that theresulting compound or construct is a fusion protein or fusionpolypeptide.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said ISV is a Nanobody®, a V_(HH), a humanized V_(HH),or a camelized V_(H).

The first building block, ISV, such as e.g. a Nanobody, or VHH of theinvention has an affinity for its—the first—target, i.e. CD4 andpolymorphic variants. The first building block, ISV or Nanobody of theinvention may for example be directed against an antigenic determinant,epitope, part, domain, subunit or confirmation (where applicable) ofsaid first target. The first building block, e.g. the first ISV, such ase.g. a Nanobody, or VHH, is chosen for its affinity for its target perse, disregarding the influence of any avidity effects. Preferred firstbuilding blocks are depicted in Table A-2(B).

“CD4” or “T-cell surface glycoprotein CD4” means the mature, native,membrane-bound CD4 protein comprising a cytoplasmic domain, ahydrophobic transmembrane domain, and an extracellular domain whichbinds to the HIV-1 gp120 envelope glycoprotein. CD4 is also known asT-cell surface antigen T4/Leu-3. Preferably CD4 is human CD4, preferablyrepresented by Uniprot accession number P01730-1 (OMIM: 186940), forinstance as represented by the amino acid sequence:

(SEQ ID NO: 1) MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSE KKTCQCPHRFQKTCSPI.

As noted before, HIV infection is characterized by a decline in thenumber of CD4⁺ T-cells in the infected person. The CD4 count of ahealthy adult/adolescent ranges from 500 cells/ml to 1,200 cells/ml. TheCD4 count measures the number of CD4 cells in a sample of blood. HIVinfection reduces the number cells comprising CD4. A very low CD4 count(less than 200 cells/mm³) is one of the ways to determine whether aperson living with HIV has progressed to stage 3 infection (AIDS).

The present invention relates to a polypeptide as described herein,wherein said polypeptide has an on rate constant (Kon) to said CD4selected from the group consisting of at least about 10² M⁻¹ s⁻¹, atleast about 10³ M⁻¹ s⁻¹, at least about 10⁴ M⁻¹ s⁻¹, at least about 10⁵M⁻¹ s⁻¹, at least about 10⁶ M⁻¹ s⁻¹, 10⁷ M⁻¹ s⁻¹, at least about 10⁸ M⁻¹s⁻¹, at least about 10⁹ M⁻¹ s⁻¹, and at least about 10¹⁰ M⁻¹ s⁻¹,preferably as measured by surface plasmon resonance

The present invention relates to a polypeptide as described herein,wherein said polypeptide has an off rate constant (Koff) to said CD4selected from the group consisting of at most about 10⁻³ s⁻¹, at mostabout 10⁻⁴ s⁻¹, at most about 10⁻⁵ s⁻¹, at most about 10⁻⁶ s⁻¹, at mostabout 10⁻⁷ s⁻¹, at most about 10⁻⁸ s⁻¹, at most about 10⁻⁹ s⁻¹, and atmost about 10⁻¹⁰ s⁻¹, preferably as measured by surface plasmonresonance.

The present invention relates to a polypeptide as described herein,wherein said polypeptide has a dissociation constant (K_(D)) to said CD4selected from the group consisting of: at most about 10⁻⁷ M, at mostabout 10⁻⁸ M, at most about 10⁻⁹ M, at most about 10⁻¹⁰ M, at most about10⁻¹¹ M, and at most about 10⁻¹² M, preferably as measured by surfaceplasmon resonance.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV binds to CD4 with an average KD value ofbetween 10 nM and 0.1 pM, such as at an average KD value of 10 nM orless, even more preferably at an average KD value of 9 nM or less, suchas less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as lessthan 400, 300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,0.5 pM, or even less such as less than 0.4 pM, preferably measured bySPR, for instance as determined by a KinExA. Kinetic Exclusion Assay(KINEXA®) (Drake et al. 2004, Analytical Biochemistry 328: 35-43)measures binding events in solution without labeling of the bindingpartners and is based upon kinetically excluding the dissociation of acomplex.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV has a high affinity when measured as amonovalent.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said average KD is measured by surface plasmon resonance(SPR) on recombinant protein.

The present invention also relates to a polypeptide as described herein,wherein said first ISV binds to a first target on the surface of a cellwith an EC₅₀ value of between 10 nM and 0.1 pM, such as at an averageEC₅₀ value of 10 nM or less, even more preferably at an average KD valueof 9 nM or less, such as less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM oreven less, such as less than 400, 300, 200, 100, 50, 40, 30, 20, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, 0.5 pM, or even less such as less than 0.4 pM.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said average EC₅₀ is measured on cells comprising saidtarget 1 but substantially lacking said target 2.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said average KD is determined by FACS, BIACORE®, ELISA,on a monovalent first ISV, such as a Nanobody, or a polypeptidecomprising a monovalent first ISV, such as a Nanobody.

It has been shown in the examples that the KD correlates well with theEC₅₀.

It is also expected that the immunoglobulin single variable domains andpolypeptides of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of its targets; or at least to those analogs, variants,mutants, alleles, parts and fragments of the CD4, and in particularhuman CD4 that contain one or more antigenic determinants or epitopesthat are essentially the same as the antigenic determinant(s) orepitope(s) to which the immunoglobulin single variable domains andpolypeptides of the invention bind to CD4, and in particular to humanCD4. Again, in such a case, the immunoglobulin single variable domainsand polypeptides of the invention may bind to such analogs, variants,mutants, alleles, parts and fragments with an affinity and/orspecificity that are the same as, or that are different from (i.e.,higher than or lower than), the affinity and specificity with which theimmunoglobulin single variable domains of the invention bind to(wild-type) receptor, e.g. CD4.

The present invention further relates to CD4 binders comprised in thepolypeptide of the invention which do not or only minimally impair orinhibit a natural function(s) of CD4, in which CD4 functions as areceptor assisting the T-cell receptor (TCR) in communicating with anantigen-presenting cell. Using its intracellular domain, CD4 amplifiesthe signal generated by the TCR by recruiting an enzyme, the tyrosinekinase Lck, which is essential for activating many molecular componentsof the signaling cascade of an activated T-cell. Various types of Thelper cells are thereby produced. CD4 also interacts directly with MHCclass II molecules on the surface of the antigen-presenting cell usingits extracellular domain. The person skilled in the art is fullyacknowledgeable of determining and measuring the (natural) function(s)of CD4, for instance by ALPHASCREEN® assay, competition ELISA, or FACSon cells and e.g., described in the experimental part.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV inhibits a pharmacologic effect of saidfirst target by less than about 50%, such as 40%, 30%, or 20% or evenless than 10%, e.g. relative to the inhibition in the absence of saidfirst ISV.

The present invention relates to a polypeptide as described herein,wherein said polypeptide inhibits multimerisation by CD4 by less thanabout 50%, such as 40%, 30%, or 20% or even less than 10%, such as lessthan 5%.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV inhibits multimerisation of said firsttarget (with T-cell receptor) by less than about 50%, such as 40%, 30%,or 20% or even less than 10%, e.g. relative to the inhibition in theabsence of said first ISV.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV binds to an allosteric site regarding thenatural function of CD4.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV does not substantially or only marginallyinhibit a (natural) function of said first target, e.g. assisting theT-cell receptor (TCR) in communicating with an antigen-presenting celland/or recruiting tyrosine kinase Lck.

The present invention relates to a polypeptide as described herein,wherein said polypeptide inhibits recruiting Lck by CD4 by less thanabout 50%, such as 40%, 30%, or 20% or even less than 10%, such as lessthan 5%.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV inhibits signalling, e.g. recruiting Lck,by said first target by less than about 50%, such as 40%, 30%, or 20% oreven less than 10%, e.g. relative to the inhibition in the absence ofsaid first ISV.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV inhibits a (natural) function of saidfirst target by less than about 50%, such as 40%, 30%, or 20% or evenless than 10%, e.g. relative to the inhibition in the absence of saidfirst ISV.

Accordingly the present invention relates to a polypeptide as describedherein, wherein said first ISV inhibits chemotaxis by less than about50%, such as 40%, 30%, or 20% or even less than 10% in an chemotaxisassay, e.g. relative to the inhibition in the absence of said first ISV.

In a preferred embodiment, the present invention relates to apolypeptide as described herein, wherein said first ISV essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   (i) CDR1 is chosen from the group consisting of SEQ ID NOs:        82-85; and amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NOs: 82-85;    -   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs:        88-91; and amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NOs: 88-91; and    -   (iii) CDR3 is chosen from the group consisting of SEQ ID NO:        96-99 and amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NOs: 96-99;

The present invention also relates to a polypeptide as described herein,wherein said first ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which said ISV is chosen from the groupconsisting of

-   -   CDR1 is SEQ ID NO: 82, CDR2 is SEQ ID NO: 88, and CDR3 is SEQ ID        NO: 96;    -   CDR1 is SEQ ID NO: 83, CDR2 is SEQ ID NO: 89, and CDR3 is SEQ ID        NO: 97;    -   CDR1 is SEQ ID NO: 84, CDR2 is SEQ ID NO: 90, and CDR3 is SEQ ID        NO: 98; and    -   CDR1 is SEQ ID NO: 85, CDR2 is SEQ ID NO: 91, and CDR3 is SEQ ID        NO: 99.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said first ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3 respectively), in which CDR1 is SEQ ID NO: 85,CDR2 is SEQ ID NO: 91 and CDR3 is SEQ ID NO: 99.

The present invention relates to a polypeptide as described herein,wherein said first ISV is chosen from the group consisting of 01B6 (SEQID NO: 17), 01E2 (SEQ ID NO: 18), 01H12 (SEQ ID NO: 19) and 03F11 (SEQID NO: 20), preferably said first ISV is 03F11 (SEQ ID NO: 20).

As described herein, a polypeptide of the invention contains at leasttwo building blocks, such as ISVs, e.g. Nanobodies, of the invention ofwhich the second building block, ISV, e.g. Nanobody, is directed againsta second target involved in HIV infection, i.e. a co-receptor for HIVinfection, including related polymorphic variants. Preferred secondbuilding blocks are depicted in Table A-2(A).

It is also expected that the immunoglobulin single variable domains andpolypeptides of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of its targets; or at least to those analogs, variants,mutants, alleles, parts and fragments of the co-receptor, such as CXCR4,and in particular human CXCR4 that contain one or more antigenicdeterminants or epitopes that are essentially the same as the antigenicdeterminant(s) or epitope(s) to which the immunoglobulin single variabledomains and polypeptides of the invention bind to the co-receptor,preferably CXCR4 and in particular to human CXCR4. Again, in such acase, the immunoglobulin single variable domains and polypeptides of theinvention may bind to such analogs, variants, mutants, alleles, partsand fragments with an affinity and/or specificity that are the same as,or that are different from (i.e., higher than or lower than), theaffinity and specificity with which the immunoglobulin single variabledomains of the invention bind to (wild-type) co-receptor, e.g. CXCR4.

The present invention relates to a polypeptide as described herein,wherein said polypeptide has an on rate constant (Kon) to said CRselected from the group consisting of at least about 10² M⁻¹ s⁻¹, atleast about 10³ M⁻¹ s⁻¹, at least about 10⁴ M⁻¹ s⁻¹, at least about 10⁵M⁻¹ s⁻¹, at least about 10⁶ M⁻¹ s⁻¹, 10⁷ M⁻¹ s⁻¹, at least about 10⁸ M⁻¹s⁻¹, at least about 10⁹ M⁻¹ s⁻¹, and at least about 10¹⁰ M⁻¹ s⁻¹,preferably as measured by surface plasmon resonance, said CR ispreferably CXCR4.

The present invention relates to a polypeptide as described herein,wherein said polypeptide has an off rate constant (Koff) to said CRselected from the group consisting of at most about 10⁻³ s⁻¹, at mostabout 10⁻⁴ s⁻¹, at most about 10⁻⁵ s⁻¹, at most about 10⁻⁶ s⁻¹, at mostabout 10⁻⁷ s⁻¹, at most about 10⁻⁸ s⁻¹, at most about 10⁻⁹ s⁻¹, and atmost about 10⁻¹⁰ s⁻¹, preferably as measured by surface plasmonresonance, said CR is preferably CXCR4.

The present invention relates to a polypeptide as described herein,wherein said polypeptide has a dissociation constant (K_(D)) to said CRselected from the group consisting of: at most about 10⁻⁷ M, at mostabout 10⁻⁸ M, at most about 10⁻⁹ M, at most about 10⁻¹⁰ M, at most about10⁻¹¹ M, and at most about 10⁻¹² M, preferably as measured by surfaceplasmon resonance, said CR is preferably CXCR4.

The present invention relates to a polypeptide as described herein,wherein said second ISV binds to said CR with an average KD value ofbetween 10 nM and 0.1 pM, such as at an average KD value of 10 nM orless, even more preferably at an average KD value of 9 nM or less, suchas less than 8, 7, 6, 5, 4, 3, 2, 1, 0.5 nM or even less, such as lessthan 400, 300, 200, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,0.5 pM, or even less such as less than 0.4 pM, preferably measured bySPR, for instance as determined by a KinExA, said CR is preferablyCXCR4.

When, designing the polypeptides of the invention, the second buildingblock, e.g. the second ISV, can be chosen for its affinity per se,disregarding the influence of any avidity effects.

In a further aspect, the present invention relates to a polypeptide asdescribed herein, wherein said second ISV has a high affinity whenmeasured as a monovalent.

The present invention relates also to a polypeptide as described herein,wherein said average KD is determined (indirectly) by any techniqueknown in the art, such as for instance SPR, FACS, or ELISA on amonovalent second ISV.

The second ISV of the invention may for example be directed against asecond antigenic determinant, epitope, part, domain, subunit orconfirmation (where applicable) of said co-receptor, and in particularhuman CXCR4.

The second target of the invention can be any target, such as a cellularreceptor, on the surface of a cell which is known to participate as aco-receptor in HIV entry.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said second ISV inhibits binding of HIV to said secondtarget, such as e.g. CR, e.g. CXCR4, by about 10%, 20%, 30%, 40%, 50%,60%, 80%, 90% and preferably 95% or even 100%, e.g. relative to theinhibition in the absence of said second ISV.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said second ISV inhibits the pharmacologic effect of HIVinfection, such as e.g. entry of HIV into a target cell, replication ofHIV, HIV reverse transcriptase activity, HIV-induced cell death, and/orHIV-induced cell-cell syncytia formation, in which said second target isinvolved by about 20%, 30%, 40%, 50%, 60%, 80%, 90% and preferably 95%or even 100%, e.g. relative to the pharmacologic effect in the absenceof said second ISV.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said second ISV displaces about 20%, 30%, 40%, 50%, 60%,80%, 90% and preferably 95% or more of HIV to said co-receptor, e.g.relative to the displacement in the absence of said second ISV.

Preferably, the polypeptide when bound impairs or inhibits HIVinfection. In a preferred embodiment the function of said second targetand or said second target is not or only minimally impaired upon bindingof the bispecific polypeptide. Consequently, binding of the bispecificpolypeptide results in limited or negligible side-effects and/ortoxicity.

As used herein, the co-receptor includes, without limitation,extracellular portions of the co-receptor capable of binding the HIVenvelope protein. The person skilled in the art will appreciate thatmore information on the sequence, function and ligands of co-receptorsfor HIV infection, such as, e.g. CXCR4, CCR5, CCR1, CCR2, CCR3, CCR8,CX3CR1, FPRL1, GPR1, GPR15, APJ, STRL33 and D6 can be found via the OMIMand Uniprot websites. The specific OMIM and Uniprot accession numbersare provided in the table below.

Receptor OMIM Uniprot CXCR4 162643 P61073 CCR5 601373 P51681 CCR1 601159P32246 CCR2 601267 P41597 CCR3 601268 P51677 CCR8 601834 P51685 CX3CR1601470 P49238 CXCR6 605163 O00574 FPRL1 136538 P25090 GPR1 600239 P46091GPR15 601166 P49685 APJ 600052 P35414 D6 602648 O00590

The present invention relates to a polypeptide as described herein,wherein said CR is chosen from the group consisting of CXCR4, CCR5,CCR1, CCR2, CCR3, CCR8, CX3CR1, CXCR6, FPRL1, GPR1, GPR15, APJ, STRL33and D6, as well as polymorphic variants thereof. Preferably saidco-receptor is CXC chemokine receptor 4 (CXCR4). The co-receptor ispreferably a human co-receptor, preferably human CXCR4.

The C-C chemokine receptor type 5, also known as CCR5 or CD195, is aprotein on the surface of white blood cells that is involved in theimmune system as it acts as a receptor for chemokines. The naturalligands for this receptor, RANTES, MIP-1β, and MIP-1α. Preferably, theCCR5 amino acid sequence is represented by:

(SEQ ID NO: 3) MDYQVSSPIYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILINCKRLKSMIDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFSGIFFIILLTIDRYLAVVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVYFLFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRST GEQEISVGL.

CCR1 has also been designated CD191 (cluster of differentiation 191).The ligands of this receptor include macrophage inflammatory protein 1alpha (MIP-1 alpha), regulated on activation normal T expressed andsecreted protein (RANTES), monocyte chemoattractant protein 3 (MCP-3),and myeloid progenitor inhibitory factor-1 (MPIF-1).

C-C chemokine receptor type 2 (CCR2 or CD192). CCR2 has two predominantforms, CC CKR2A and CC CKR2B. CCR2 is the receptor for CCL2, thepredominant agonist for CC CKR2A is MCP1, while both MCP1 and MCP3 areligands for the CC CKR2B isoform.

C-C chemokine receptor type 3 (CCR3) is a protein that in humans isencoded by the CCR3 gene (also designated as CD193). This receptor bindsand responds to a variety of chemokines, including eotaxin (CCL11),eotaxin-3 (CCL26), MCP-3 (CCL7), MCP-4 (CCL13), and RANTES (CCL5).

Chemokine (C-C motif) receptor 8, also known as CCR8, is a protein whichin humans is encoded by the CCR8 gene. CCR8 has also been designatedCDw198. The ligand of the CCR8 is CCL1. CCL8 also functions as a CCR8agonist.

CX3C chemokine receptor 1 (CX3CR1) also known as the fractalkinereceptor or G-protein coupled receptor 13 (GPR13) is a protein that inhumans is encoded by the CX3CR1 gene. This receptor binds the chemokineCX3CL1 (also called neurotactin or fractalkine).

C-X-C chemokine receptor type 6 (CXCR6) is a protein that in humans isencoded by the CXCR6 gene. CXCR6 has also been designated CD186 (clusterof differentiation 186) and STRL33. STRL33 is expressed in lymphoidtissues and activated T-cells, and is induced in activated peripheralblood lymphocytes. STRL33 is a receptor for one of the chemokines. Othernames for this receptor are Bonzo and TYMSTR.

FPRL1: N-formyl peptide receptor 2 is a G-protein coupled receptor(GPCR) protein that in humans is encoded by the FPR2 gene.

GPR1 is a member of the G protein-coupled receptor family oftransmembrane receptors. It functions as a receptor for chemerin.

G-protein coupled receptor 15 (GPR15) is a protein that in humans isencoded by the GPR15 gene an orphan heterotrimeric guaninenucleotide-binding protein (G protein)-coupled receptor.

The apelin receptor (also known as the APJ receptor) is a Gprotein-coupled receptor which binds apelin and Apela/ELABELA/Toddler.

D6 or Chemokine-binding protein 2 is a protein that in humans is encodedby the CCBP2 gene. CXCR4 is also known as fusin or CD184. A summary ofCXCR4 function is provided, for instance, in Steen et al. (TargetingCXCR4 in HIV Cell-Entry Inhibition, Mini-Reviews in Medicinal Chemistry,2009, 9, 1605-1621). The function of CXCR4 is regulated predominantly bythe interaction of CXCR4 with its natural ligand Stromal Cell DerivedFactor-1 (SDF-1), also called CXCL12 (chemokine C-X-C motif ligand 12.However, MIF can also function as a ligand for CXCR4 (See e.g., Schwartzet al., FEBS Lett 2009, 583: 2749; Bernhagen et al., Nat. Med. 2007, 13:587). SDF-1 is found in two forms, SDF-1α/CXC12a and SDF-1β/CXC12b,which are produced by alternate splicing of the two genes. CXCR4 is morebroadly expressed than most other chemokine receptors and was for a longtime thought to be strictly monogamous in its relationship with thenatural ligand, SDF-1. However, CXCR7 also uses SDF-1 as a ligand, andrecent evidence has emerged that ubiquitin can also function as a ligandof CXCR4 (Saini V et al., (2010). J. Biol. Chem. 285 (20): 15566-76).The CXCR4/SDF-1 axis is involved in immune cell trafficking as itregulates chemotaxis of B cells, plasma cells, CD4⁺ T-cells, anddendritic cells in vivo and activates the tight adhesion of rollingT-cells on activated epithelial cells and their subsequenttransendothelial migration. The CXCR4/SDF-1 axis is required for normalmyelopoiesis and lymphopoiesis and is in addition critical for properembryonic development of numerous organ systems. CXCR4 is expressed innumerous tissues, such as peripheral blood leukocytes, spleen, thymus,spinal cord, heart, placenta, lung, liver, skeletal muscle, kidneypancreas, cerebellum, cerebral cortex and medulla, brain microvascular,coronary artery and umbilical cord endothelial cells. Mice lacking CXCR4or SDF-1 have impaired hematopoiesis, derailed cerebellar neuronmigration, defective formation of large vessels and cardiac ventricularseptal defects, which can lead to cardiac failure (See e.g., Ma et al.,PNAS 1998, 95:9448-9453; Zou et al., Nature 1998, 393:595-599).Preferably, the CXCR4 amino acid sequence is represented by:

(SEQ ID NO: 2) MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS.

While CXCR4, CCR5, CCR1, CCR2, CCR3, CCR8, CX3CR1, FPRL1, GPR1, GPR15,APJ, STRL33 and D6 can be used by HIV as a co-receptor to enter a cell,the “natural” function of CXCR4, CCR5, CCR1, CCR2, CCR3, CCR8, CX3CR1,FPRL1, GPR1, GPR15, APJ, STRL33 and D6 is in chemokine signaling. A“natural” function of the co-receptor (second target) relates to anychange in a measurable biological or biochemical property elicited bysaid co-receptor (in the absence of HIV binding), includingphysiological changes of the cell such as changes in proliferation,differentiation, migration, survival, apoptosis, transport processes,metabolism, motility, cytokine release, cytokine composition, secondmessengers, enzymes, receptors, etc. due to chemokine signalling.Preferably the function of a target is determined by cell culture basedpotency assays as well known in the art. The “natural” function(s) ofthe co-receptor can be determined by any suitable assay known by theperson skilled in the art, such as ELISA, FACS, Scatchard analysis,ALPHASCREEN®, SPR, functional assays, etc., for instance as discussedherein.

The efficacy or potency of the immunoglobulin single variable domainsand polypeptides of the invention, and of compositions comprising thesame on the natural function on said CR, can be tested using anysuitable in vitro assay, cell-based assay, in vivo assay and/or animalmodel known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example include liganddisplacement assays (Burgess et al., Cancer Res 2006 66:1721-9),dimerization assays (WO2009/007427A2, Goetsch, 2009), signaling assays(Burgess et al., Mol Cancer Ther 9:400-9), proliferation/survival assays(Pacchiana et al., J Biol Chem 2010 Sep M110.134031), cell adhesionassays (Holt et al., Haematologica 2005 90:479-88) and migration assays(Kong-Beltran et al., Cancer Cell 6:75-84), endothelial cell sproutingassays (Wang et al., J Immunol. 2009; 183:3204-11), and in vivoxenograft models (Jin et al., Cancer Res. 2008 68:4360-8), as well asthe assays and animal models used in the experimental part below and inthe prior art cited herein. A means to express the inhibition of saidsecond target is by IC₅₀.

In one aspect, the disclosure provides an immunoglobulin single variabledomain that inhibits binding of HIV to a co-receptor, preferably CXCR4,and does not displace a natural ligand from this co-receptor.

The present invention relates to a polypeptide according to any of thepreceding claims, wherein said polypeptide inhibits binding of a naturalligand to said CR by less than about 50%, such as 40%, 30%, or 20% oreven less than 10%, such as less than 5%.

In some embodiments, the natural ligand is Stromal Cell-Derived Factor-1beta (SDF-13) or Stromal Cell-Derived Factor-1 alpha (SDF-1α). In someembodiments, the IC₅₀ of SDF-1α or SDF-1β displacement from CXCR4 in thepresence of the polypeptide of the invention is 10 nM or higher. In someembodiments, the IC₅₀ of SDF-1α or SDF-1β displacement from CXCR4 in thepresence of the immunoglobulin single variable domain is 250 nM orhigher. In some embodiments, the IC₅₀ of SDF-1α or SDF-1β displacementfrom CXCR4 in the presence of the polypeptide of the invention is 1 pMor higher. In some embodiments, the IC₅₀ of SDF-1α or SDF-1βdisplacement from CXCR4 in the presence of the immunoglobulin singlevariable domain is greater than the IC₅₀ of HIV inhibition.

In some embodiments, the IC₅₀ of HIV inhibition is lower than 50 nM,lower than 10 nM, or lower than 1 nM. In some embodiments the IC₅₀ ofSDF-1α or SDF-1β displacement from CXCR4 in the presence of thepolypeptide is greater than the IC₅₀ of HIV inhibition.

In some embodiments, the IC₅₀ of displacement of SDF-1α or SDF-1β fromCXCR4 in the presence of the immunoglobulin single variable domain orpolypeptide construct thereof is greater than the IC₅₀ of the inhibitionof binding of HIV to the CR, such as CXCR4 by the immunoglobulin singlevariable domain or polypeptide constructs thereof by 1 pM or more, 10 pMor more, 100 pM or more, 500 pM or more, 1 nM or more, 10 nM or more, 20nM or more, 30 nM or more, 40 nM or more, 50 nM or more, 60 nM or more,70 nM or more, 80 nM or more, 100 nM or more, 500 nM or more, 1 μM ormore, 10 μM or more, 50 μM or more, 100 μM or more, up to 1 mM.

In some embodiments, the IC₅₀ of displacement of SDF-1α or SDF-1β fromCXCR4 in the presence of the immunoglobulin single variable domain orpolypeptide thereof is greater than the IC₅₀ of the inhibition ofbinding of HIV to CXCR4 by the immunoglobulin single variable domain orpolypeptide thereof by 1% or more, 2% or more, 5% or more, 10% or more,20% or more, 50% or more, 100% or more, 2× higher or more, 5× higher ormore, 10× higher or more, 20× higher or more, 50× higher or more, 100×higher or more, 1000× higher or more, up to 10,000× higher or more.

In some embodiments, the inhibition of HIV binding to CD4 and/or aco-receptor such as e.g. CXCR4 by the polypeptide of the invention isstronger (e.g., has a lower IC₅₀) than the inhibition of HIV binding toCXCR4 by AMD3100.

In some embodiments, the inhibition of HIV binding to CD4 and/or aco-receptor such as e.g. CXCR4 by the polypeptide of the invention isstronger (e.g., has a lower IC₅₀) than the inhibition of HIV binding toCXCR4 by 283D2-20GS-283D4 (see WO2009/138519).

Since various cell surface receptors require dimerization foractivation, it is preferred that in such cases the second ISV of theinvention does not impair these dimerization sites.

In some embodiments, the immunoglobulin single variable domains andpolypeptides thereof do not displace a natural ligand from theco-receptor, e.g. CXCR4. In some embodiments, the natural ligand isStromal Cell-Derived Factor-1(3 (SDF-13) or Stromal Cell-DerivedFactor-1α (SDF-1α).

Displacing, as used herein, includes both complete and partialdisplacement. Thus, the disclosure embraces immunoglobulin singlevariable domains and polypeptides comprising one or more immunoglobulinsingle variable domains that do not displace natural ligand, or thatdisplace less than 1%, less than 2%, less than 5%, less than 10%, lessthan 20%, less than 30%, less than 40%, less than 50%, less than 60%,less than 70%, less than 80%, less than 90%, up to less than 99% ofdisplacement of a natural ligand from the CR, e.g. CXCR4. In someembodiments, the IC₅₀ of displacement of the natural ligand from the CR,such as e.g. SDF-1α or SDF-1β from CXCR4, in the presence of theimmunoglobulin single variable domain or polypeptide thereof is 1 pM orhigher, 10 pM or higher, 100 pM or higher, 500 pM or higher, 1 nM orhigher, 10 nM or higher, 20 nM or higher, 30 nM or higher, 40 nM orhigher, 50 nM or higher, 60 nM or higher, 70 nM or higher, 80 nM orhigher, 100 nM or higher, 500 nM or higher, 1 μM or higher, 10 μM orhigher, 50 μM or higher, 100 μM or higher, up to 1 mM.

The present invention also relates to a polypeptide as described herein,wherein said second ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which

-   -   (i) CDR1 is chosen from the group consisting of SEQ ID NOs:        34-40; and amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NOs: 34-408;    -   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs:        48-56; and amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NOs: 48-56; and    -   (iii) CDR3 is chosen from the group consisting of SEQ ID NO:        67-75 and amino acid sequences that have 1, 2, 3 or 4 amino acid        difference(s) with SEQ ID NOs: 67-75.

The present invention also relates to a polypeptide as described herein,wherein said second ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which said ISV is chosen from the groupconsisting of

-   -   CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID        NO: 67;    -   CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID        NO: 68;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID        NO: 69;    -   CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID        NO: 70;    -   CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID        NO: 71;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID        NO: 72;    -   CDR1 is SEQ ID NO: 38, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID        NO: 73;    -   CDR1 is SEQ ID NO: 39, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID        NO: 74; and    -   CDR1 is SEQ ID NO: 40, CDR2 is SEQ ID NO: 56, and CDR3 is SEQ ID        NO: 75.

Accordingly, the present invention relates to a polypeptide as describedherein, in which said second ISV essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3 respectively), in which CDR1 is SEQ ID NO: 35,CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 69.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said second ISV is chosen from the group consisting of238D4 (SEQ ID NO: 4), 281A5 (SEQ ID NO: 5), 281E10 (SEQ ID NO: 6), 281D4(SEQ ID NO: 7), 281A6 (SEQ ID NO: 8), 281F12 (SEQ ID NO: 9), 283B6 (SEQID NO: 10), 283E2 (SEQ ID NO: 11), 283F1 (SEQ ID NO: 12), 15F5 (SEQ IDNO: 13), 15G11 (SEQ ID NO: 14), 15A1 (SEQ ID NO: 15) and 10C3 (SEQ IDNO: 16), preferably in which said second ISV is 281F12 (SEQ ID NO: 9).

In an embodiment, the present invention relates to a polypeptidecomprising a first and a second immunoglobulin single variable domain(ISV), wherein said first ISV binds to CD4 and/or polymorphic variantspresent on the surface of a cell; said second ISV binds to a co-receptor(CR) present on the surface of said cell, preferably wherein said CR ischosen from the group consisting of CXCR4, CCR5, CCR1, CCR2, CCR3, CCR8,CX3CR1, CXCR6, FPRL1, GPR1, GPR15, APJ, and D6 and related polymorphicvariants. Preferably said CR is CXCR4.

In a preferred embodiment, the present invention relates to apolypeptide as described herein, wherein said first ISV essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 82-85;    and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 82-85;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 88-91;    and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 88-91; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 96-99    and amino acid sequences that have 1, 2, 3 or 4 amino acid    difference(s) with SEQ ID NOs: 96-99;

and, wherein said second ISV essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3 respectively), in which

-   (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 34-40;    and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 34-40;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 48-56;    and amino acid sequences that have 1, 2 or 3 amino acid    difference(s) with SEQ ID NOs: 48-56; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 67-75    and amino acid sequences that have 1, 2, 3 or 4 amino acid    difference(s) with SEQ ID NOs: 67-75.

The present invention also relates to a polypeptide as described herein,wherein said first ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which said ISV is chosen from the groupconsisting of

-   -   CDR1 is SEQ ID NO: 82, CDR2 is SEQ ID NO: 88, and CDR3 is SEQ ID        NO: 96;    -   CDR1 is SEQ ID NO: 83, CDR2 is SEQ ID NO: 89, and CDR3 is SEQ ID        NO: 97;    -   CDR1 is SEQ ID NO: 84, CDR2 is SEQ ID NO: 90, and CDR3 is SEQ ID        NO: 98; and    -   CDR1 is SEQ ID NO: 85, CDR2 is SEQ ID NO: 91, and CDR3 is SEQ ID        NO: 99.

wherein said second ISV essentially consists of 4 framework regions (FR1to FR4, respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which said ISV is chosen from the groupconsisting of

-   -   CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID        NO: 67;    -   CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID        NO: 68;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID        NO: 69;    -   CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID        NO: 70;    -   CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID        NO: 71;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID        NO: 72;    -   CDR1 is SEQ ID NO: 38, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID        NO: 73;    -   CDR1 is SEQ ID NO: 39, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID        NO: 74; and    -   CDR1 is SEQ ID NO: 40, CDR2 is SEQ ID NO: 56, and CDR3 is SEQ ID        NO: 75.

In a preferred embodiment, the present invention relates to apolypeptide as described herein, wherein said first ISV essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   (i) CDR1 is chosen from the group consisting of SEQ ID NO: 85 and    amino acid sequences that have 1, 2 or 3 amino acid difference(s)    with SEQ ID NO: 85;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NO: 91 and    amino acid sequences that have 1, 2 or 3 amino acid difference(s)    with SEQ ID NO: 91; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 99 and    amino acid sequences that have 1, 2, 3 or 4 amino acid difference(s)    with SEQ ID NO: 99;

and, wherein said second ISV essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3 respectively), in which

-   (i) CDR1 is chosen from the group consisting of SEQ ID NO: 35 and    amino acid sequences that have 1, 2 or 3 amino acid difference(s)    with SEQ ID NO: 35;-   (ii) CDR2 is chosen from the group consisting of SEQ ID NO: 50 and    amino acid sequences that have 1, 2 or 3 amino acid difference(s)    with SEQ ID NO: 50; and-   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: 69 and    amino acid sequences that have 1, 2, 3 or 4 amino acid difference(s)    with SEQ ID NO: 69.

In a preferred embodiment, the present invention relates to apolypeptide as described herein, wherein said first ISV essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   (i) CDR1 is represented by SEQ ID NO: 85;-   (ii) CDR2 is represented by SEQ ID NO: 91; and-   (iii) CDR3 is represented by SEQ ID NO: 99;

and, wherein said second ISV essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3 respectively), in which

-   (i) CDR1 is represented by SEQ ID NO: 35;-   (ii) CDR2 is represented by SEQ ID NO: 50; and-   (iii) CDR3 is represented by SEQ ID NO: 69.

In a preferred embodiment, the present invention relates to apolypeptide as described herein, wherein said first ISV is chosen fromthe group consisting of 01B6 (SEQ ID NO: 17), 01E2 (SEQ ID NO: 18),01H12 (SEQ ID NO: 19) and 03F11 (SEQ ID NO: 20), and wherein said secondISV is chosen from the group consisting of 238D4 (SEQ ID NO: 4), 281A5(SEQ ID NO: 5), 281E10 (SEQ ID NO: 6), 281D4 (SEQ ID NO: 7), 281A6 (SEQID NO: 8), 281F12 (SEQ ID NO: 9), 283B6 (SEQ ID NO: 10), 283E2 (SEQ IDNO: 11), 283F1 (SEQ ID NO: 12), 15F5 (SEQ ID NO: 13), 15G11 (SEQ ID NO:14), 15A1 (SEQ ID NO: 15) and 10C3 (SEQ ID NO: 16).

In a preferred embodiment, the present invention relates to apolypeptide chosen from the group consisting of 03F11-9GS-281F12 (SEQ IDNO: 101), 03F11-25GS-281F12 (SEQ ID NO: 102), 03F11-35GS-281F12 (SEQ IDNO: 103), 281F12-9GS-03F11 (SEQ ID NO: 104), 281F12-25GS-03F11 (SEQ IDNO: 105), 281F12-35GS-03F11 (SEQ ID NO: 106),15G11(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 107),15F05(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 108), and281F12(Q108L)-15GS-ALB11-15GS-03F11(Q108L) (SEQ ID NO: 109).

In a specific, but non-limiting aspect of the invention, which will befurther described herein, the polypeptides of the invention have anincreased half-life in serum (as further described herein) compared tothe immunoglobulin single variable domain from which they have beenderived. For example, an immunoglobulin single variable domain of theinvention may be linked (chemically or otherwise) to one or more groupsor moieties that extend the half-life (such as PEG), so as to provide aderivative of an amino acid sequence of the invention with increasedhalf-life.

As demonstrated in the examples, half-life extension did not affectpotency substantially. This indicates that half-life extended bispecificconstructs are still capable of binding simultaneously to theirrespective targets.

In a specific aspect of the invention, a compound of the invention or apolypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise immunoglobulin single variable domainsor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);immunoglobulin single variable domains of the invention that comprise atleast one additional binding site for binding to a serum protein (suchas serum albumin); or polypeptides of the invention which comprise atleast one amino acid sequence of the invention that is linked to atleast one moiety (and in particular at least one amino acid sequence)which increases the half-life of the amino acid sequence of theinvention. Examples of polypeptides of the invention which comprise suchhalf-life extending moieties or immunoglobulin single variable domainswill become clear to the skilled person based on the further disclosureherein; and for example include, without limitation, polypeptides inwhich the one or more immunoglobulin single variable domains of theinvention are suitably linked to one or more serum proteins or fragmentsthereof (such as (human) serum albumin or suitable fragments thereof) orto one or more binding units that can bind to serum proteins (such as,for example, domain antibodies, immunoglobulin single variable domainsthat are suitable for use as a domain antibody, single domainantibodies, immunoglobulin single variable domains that are suitable foruse as a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies that can bindto serum proteins such as serum albumin (such as human serum albumin),serum immunoglobulins such as IgG, or transferrin; reference is made tothe further description and references mentioned herein); polypeptidesin which an amino acid sequence of the invention is linked to an Fcportion (such as a human Fc) or a suitable part or fragment thereof; orpolypeptides in which the one or more immunoglobulin single variabledomains of the invention are suitable linked to one or more smallproteins or peptides that can bind to serum proteins, such as, withoutlimitation, the proteins and peptides described in WO 91/01743, WO01/45746, WO 02/076489, WO2008/068280, WO2009/127691 andPCT/EP2011/051559.

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life e.g., in humans that is increased with more than 1hours, preferably more than 2 hours, more preferably more than 6 hours,such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding amino acid sequence of the invention perse.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life e.g. in humansthat is increased with more than 1 hours, preferably more than 2 hours,more preferably more than 6 hours, such as more than 12 hours, or evenmore than 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In a particularly preferred but non-limiting aspect of the invention,the invention provides a polypeptide of the invention comprising a firstand a second immunoglobulin single variable domain (ISV); and furthercomprising one or more (preferably one) serum albumin bindingimmunoglobulin single variable domain as described herein, e.g. theserum albumin binding immunoglobulin single variable domain of Alb8,Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA,Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (cf. Table HLE Below).

TABLE HLE Name SEQ ID NO Amino acid sequence Alb8 111EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 112EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 113EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 114EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 115EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11 116EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL (S112K)-AYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 117EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 118EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 119EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 120EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 121EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GG 122EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 123EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG

Accordingly, the present invention relates to a polypeptide as describedherein, further comprising a serum protein binding moiety.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety binds serum albumin.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety is an immunoglobulin singlevariable domain binding serum albumin.

The present invention relates to a polypeptide as described herein,wherein said ISV binding serum albumin essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which CDR1 is SFGMS(SEQ ID NO: 124), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 125), and inwhich CDR3 is GGSLSR (SEQ ID NO: 126).

The present invention relates to a polypeptide as described herein,wherein said ISV binding serum albumin comprises Alb8, Alb23, Alb129,Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA,Alb82-G, Alb82-GG, Alb82-GGG.

In the polypeptides of the invention, the two or more building blocks,ISVs, such as e.g. Nanobodies, and the optionally one or morepolypeptides one or more other groups, drugs, agents, residues, moietiesor binding units may be directly linked to each other (as for exampledescribed in WO 99/23221) and/or may be linked to each other via one ormore suitable spacers or linkers, or any combination thereof. Suitablespacers or linkers for use in multivalent and multispecific polypeptideswill be clear to the skilled person, and may generally be any linker orspacer used in the art to link amino acid sequences. Preferably, saidlinker or spacer is suitable for use in constructing proteins orpolypeptides that are intended for pharmaceutical use. In contrast toexpectations, there was no apparent effect of linker length between thefirst ISV and second ISV as demonstrated in the examples.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678). Preferred linkers are depicted in Table Linkers below.

Table Linkers Name SEQ ID NO Amino acid sequence 5GS 127 GGGGS 7GS 128SGGSGGS 9GS 129 GGGGSGGGS 10GS 130 GGGGSGGGGS 15GS 131 GGGGSGGGGSGGGGS18GS 132 GGGGSGGGGSGGGGGGGS 20G5 133 GGGGSGGGGSGGGGSGGGGS 25G5 134GGGGSGGGGSGGGGSGGGGSGGGGS 30G5 135 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35G5136 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for a chemokine, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that comprisebuilding blocks, ISVs or Nanobodies directed against a first and secondtarget, the length and flexibility of the linker are preferably suchthat it allows each building block, ISV or Nanobody of the inventionpresent in the polypeptide to bind to its cognate target, e.g. theantigenic determinant on each of the targets.

Again, based on the disclosure herein, the skilled person will be ableto determine the optimal linker(s) for use in a specific polypeptide ofthe invention, optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues can provide improved hydrophilic properties, whereaslinkers that form or contain small epitopes or tags can be used for thepurposes of detection, identification and/or purification. Again, basedon the disclosure herein, the skilled person will be able to determinethe optimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more building blocks, ISV orNanobodies, it is possible to link them by use of a linker with three ormore “arms”, which each “arm” being linked to a building block, ISV orNanobody, so as to provide a “star-shaped” construct. It is alsopossible, although usually less preferred, to use circular constructs.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said first ISV and said second ISV and possibly said ISVbinding serum albumin are directly linked to each other or are linkedvia a linker.

The present invention relates to a polypeptide as described herein,wherein said linker is chosen from the group consisting of linkers of5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS and 30GS.

The present invention relates to a polypeptide as described herein,wherein said serum protein binding moiety is a non-antibody basedpolypeptide (e.g. PEG).

In the present, medical context, HIV infection is the tendency of amedical condition to become progressively worse and if not treatedresults in AIDS and potentially in death. HIV infection results in adecline in the number of CD4⁺ T-cells in the infected person. Below acritical number of CD4⁺ T-cells, cell-mediated immunity is effectivelylost, and a variety of infections by opportunistic micro-organismsappear, resulting in Acquired Immunodeficiency Syndrome (AIDS). Thesephenomena of (progressing) HIV infection are well known in the art.

The pharmacologic effect of the polypeptides of the invention thereforewill reside eventually in inhibiting or impairing at least one, butpreferably more than one result of an HIV infection.

In one aspect, the disclosure provides methods for lowering theHIV-titer (e.g. viral load) in a subject, the method comprisingadministering to the subject a therapeutically effective amount of animmunoglobulin single variable domain or a polypeptide comprising one ormore immunoglobulin single variable domains of the invention to lowerthe HIV-titer in the subject. In some embodiments, the administeredimmunoglobulin single variable domain or polypeptide thereof inhibitsbinding of HIV to CD4 and/or a CR, such as e.g. CXCR4 receptor butpreferably does not displace a natural ligand from said CR, such as e.g.CXCR4. In some embodiments, the administered immunoglobulin singlevariable domain or polypeptide thereof have only minimal undesirableside effects.

The immunoglobulin single variable domains and polypeptides thereofinhibit binding of HIV to CD4 and/or a CR, such as e.g. CXCR4. Bybinding the CD4 and/or a CR, such as e.g. CXCR4 the immunoglobulinsingle variable domain or polypeptide thereof prevents HIV from enteringthe cell. HIV cannot survive for prolonged periods of time outside of acellular environment. Thus, if HIV cannot enter the cell and remains inthe extracellular environment, HIV will eventually expire and bedisposed of by the body, eventually resulting in the lowering of theHIV-titer in a person.

Methods for determining the amount of HIV in a subject (the HIV-titer)are routine in the art (cf. supra). Generally, a blood sample from asubject is provided and the amount of HIV (e.g., the amount ofHIV-particles) is determined either directly (by assaying for thepresence of HIV) or indirectly (e.g., by assaying for the presence ofantibodies against HIV). Determining the presence of HIV, or antibodiesagainst HIV, is routine in the art and can be performed, for instance,by ELISA. Additional methods of determining the amount of HIV in asubject include the functional inhibition assays discussed above, assaysto determine the presence and amount of specific antigens, such as thep24 antigen test (commercially available for instance throughPerkinElmer, and Advanced Bioscience Laboratories), and assays todetermine the presence and amount of specific nucleic acids encoding theHIV genome, for instance through reverse transcriptase activity (e.g.,ExaVir Load; Cavidi Tech-AB, Uppsala, Sweden; See e.g., Sivapalasingamet al., J Clin Microbiol 2005, 43, 3793).

The methods disclosed herein are applicable to any form of HIV includingHIV-1 and HIV-2, and all of the subclasses, e.g., HIV-1B, HIV-1D, etc.In some embodiments, the methods disclosed herein are applicable toviruses related to HIV, such as the simian virus SIV.

The present invention relates to a polypeptide as described herein, foruse in treating a subject in need thereof (infected with HIV, preferablyHIV-1, preferably subtype C).

The present invention relates to a pharmaceutical composition comprisinga polypeptide as described herein.

The present invention relates to a method for delivering a prophylacticand/or therapeutic polypeptide to a specific location, tissue or celltype in the body, the method comprising the steps of administering to asubject a polypeptide as described herein.

The present invention relates to a method for treating a subject in needthereof comprising administering a polypeptide as described herein.

Also, the present invention relates to a method for treating a subjectas described above, wherein said subject is infected with HIV R5, HIVX4, and/or HIV X4R5.

In one aspect, the disclosure provides a method for treating a subjectsuffering from infection by HIV, the method comprising administering tothe subject an immunoglobulin single variable domain or a polypeptideconstruct comprising one or more immunoglobulin single variable domainsto treat the infection by HIV. In some embodiments, the administeredimmunoglobulin single variable domain or polypeptide constructs thereofinhibits binding of HIV to CD4 and/or a CR, such as e.g. CXCR4 butpreferably does not displace a natural ligand from said CR, such as e.g.CXCR4. In some embodiments, the administered immunoglobulin singlevariable domain or polypeptides construct thereof have only minimalundesirable side effects.

The term “treating a subject suffering from HIV infection” as usedherein refers to any method that results in a reduction in the severityof symptoms (e.g., opportunistic microbial infections) or number ofsymptoms (e.g., number of opportunistic infections) associated with HIVinfection in a subject.

In some embodiments, treating a subject suffering from HIV results in anincrease in the white blood cell count in said subject.

In some embodiments, treating a subject suffering from HIV results in anincrease in the number of CD4⁺ T-cells in the subject.

In some embodiments, treating a subject suffering from HIV results in adecrease in the HIV-titer in the subject.

Accordingly, the present invention relates to a method for lowering theHIV-titer in a subject, the method comprising administering to thesubject a therapeutically effective amount of a polypeptide as describedherein to lower the HIV-titer in the subject.

In some embodiments, treating a subject suffering from HIV results in adecrease in the number of opportunistic microbial infections in theperson.

Whether treatment is effective can be assessed, for instance, bydetermining the change in one or more physiological parametersassociated with HIV infection (e.g., lowering of HIV-titer, decrease ofthe number of infected cells, increase in the amount of CD4+ T-cells),or by assessing the health of a subject at the whole-body level (e.g.,decrease in the number of opportunistic infections).

In one aspect, the disclosure provides methods for treating a subjectsuffering from HIV infection with minimal unwanted side effects.Traditional anti-HIV treatment regimens (e.g., ART), including theadministration of HIV-protease inhibitors and HIV-reverse transcriptaseinhibitors is associated with a number of unwanted side effects,including liver toxicity, bloating, loss of appetite, etc. A person ofordinary skill in the art can determine if unwanted side effects havedecreased, e.g., by assessing the level of liver toxicity, bloating,etc. In some embodiments, the unwanted side effects are side effectsassociated with stem cell mobilization. Methods of determining the levelof stem cell mobilization are known in the art.

A subject, as used herein, includes a mammal that is susceptible to HIVinfection (e.g., a human), or infection by a related virus such as SIV(e.g., a monkey). In some embodiments, the subject is a primate. In someembodiment, the subject is human. In some embodiments, the subject isreceiving, or has received, one or more anti-HIV treatment regimens(e.g., ART therapy or a component thereof).

As demonstrated in the present invention, it is very difficult for HIVstrain to become resistant to the polypeptide of the invention, evenwhen using monovalent Nanobodies for inducing resistance.

Surprisingly, even on viruses that are resistant to one of thereceptors, the bispecific polypeptide retains a strong potency in thepicoMolar range in inhibition of HIV1 entry, suggesting thatfunctionality of only one of the arms of the bispecific polypeptides issufficient for the potent inhibition, when the other arm can providebinding avidity.

Accordingly, the invention relates to a method for treating a subjectinfected with HIV, comprising administering a polypeptide as describedherein, wherein said HIV does not develop, e.g. delays, resistance tosaid polypeptide for at least 3 months, such as at least 6 months, oreven longer such as e.g. 9 m, 11 m, 1 y, 1.5 y, 2 y or even longer.

In the above methods, the amino acid sequences, ISV's, such as e.g.Nanobodies, and/or polypeptides of the invention and/or the compositionscomprising the same can be administered in any suitable manner,depending on the specific pharmaceutical formulation or composition tobe used. Thus, the amino acid sequences, ISV's, such as e.g. Nanobodies,and/or polypeptides of the invention and/or the compositions comprisingthe same can for example be administered orally, intraperitoneally (e.g.intravenously, subcutaneously, intramuscularly, or via any other routeof administration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, ISV's, such as e.g. Nanobodies, and/orpolypeptides of the invention and/or the compositions comprising thesame are administered according to a regime of treatment that issuitable for preventing and/or treating the HIV infection to beprevented or treated. The clinician will generally be able to determinea suitable treatment regimen, depending on factors such as the stage ofthe HIV infection to be treated, the severity of the HIV infection to betreated and/or the severity of the symptoms thereof, the specific aminoacid sequence, ISV, such as e.g. a Nanobody, or polypeptide of theinvention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, ISV's, such as e.g. Nanobodies, and/orpolypeptides of the invention, or of one or more compositions comprisingthe same, in one or more pharmaceutically effective amounts or doses.The specific amount(s) or doses to be administered can be determined bythe clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of an HIV infectionmentioned herein and depending on the specific strain or type and stageof the disease to be treated, the potency of the specific amino acidsequence, ISV, such as e.g. a Nanobody, and polypeptide of the inventionto be used, the specific route of administration and the specificpharmaceutical formulation or composition used, the amino acidsequences, ISV's, such as e.g. Nanobodies, and polypeptides of theinvention will generally be administered in an amount between 1 gram and0.01 milligram per kg body weight per day, preferably between 0.1 gramand 0.01 milligram per kg body weight per day, such as about 0.1, 1, 10,100 or 1000 milligram per kg body weight per day, e.g. from 0.1 mg perkg to 25 mg per kg of the subject's body weight; either continuously(e.g. by infusion), as a single daily dose or as multiple divided dosesduring the day. The clinician will generally be able to determine asuitable daily dose, depending on the factors mentioned herein. It willalso be clear that in specific cases, the clinician may choose todeviate from these amounts, for example on the basis of the factorscited above and his expert judgment. Generally, some guidance on theamounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, ISV, such ase.g. a Nanobody, or polypeptide of the invention will be used. It ishowever within the scope of the invention to use two or more amino acidsequences, ISV's, such as e.g. Nanobodies, and/or polypeptides of theinvention in combination.

The ISV's, such as e.g. Nanobodies, amino acid sequences andpolypeptides of the invention may also be used in combination with oneor more further pharmaceutically active compounds or principles, i.e. asa combined treatment regimen, which may or may not lead to a synergisticeffect. Again, the clinician will be able to select such furthercompounds or principles, as well as a suitable combined treatmentregimen, based on the factors cited above and his expert judgment.

In particular, the amino acid sequences, ISV's, such as e.g. Nanobodies,and polypeptides of the invention may be used in combination with otherpharmaceutically active compounds or principles that are or can be usedfor the prevention and/or treatment of the HIV infection and/or anyopportunistic infection, disease and/or disorder cited herein, as aresult of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

In order to evade HIV resistance and prolong efficacy, contemporaryanti-HIV treatment regimens comprise a cocktail of anti-HIV drugs.Hence, it is advantageous to include the polypeptide of the inventioninto an anti-HIV treatment regimen, such as e.g. ART therapy or acomponent thereof. In some embodiments, the subject is treated with apolypeptide of the invention and ART therapy or a component thereof,such as e.g. one or more protease inhibitors (PRs), e.g. amprenavir(AMP), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir(NFV), ritonavir (RTV) or saquinavir (SQV); and/or reverse transcriptaseinhibitors (RTIs), e.g. a non-nucleoside reverse transcriptase inhibitor(NNRTI) [abacavir (ABC), delavirdine (DLV), efavirenz (EFV), nevirapine(NVP) and tenofovir (TFV)]; or a nucleoside analogue reversetranscriptase inhibitor (NRTI) [didanosine (ddl), stavudine (d4T),lamivudine (3TC) and zidovudine (ZDV)].

In some embodiments, HIV is, or has become, resistant to one or moreanti-HIV treatment regimens (e.g., ART therapy or a component thereof),for instance, wherein HIV is, or has become, resistant to one or moreprotease inhibitors (PRs), e.g. amprenavir (AMP), atazanavir (ATV),indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV) orsaquinavir (SQV); and/or reverse transcriptase inhibitors (RTIs), e.g. anon-nucleoside reverse transcriptase inhibitor (NNRTI) [abacavir (ABC),delavirdine (DLV), efavirenz (EFV), nevirapine (NVP) and tenofovir(TFV)]; or a nucleoside analogue reverse transcriptase inhibitor (NRTI)[didanosine (ddl), stavudine (d4T), lamivudine (3TC) and zidovudine(ZDV)].

The present invention also relates to a method for treating a subjectinfected with HIV, comprising administering a polypeptide as describedherein, wherein said subject is resistant against at least one otheranti-HIV agent.

In some embodiments, the subject has unwanted side effects whenreceiving one or more anti-HIV treatment regimens (e.g., ART therapy ora component thereof).

The present invention relates to a method for treating a subject asdescribed herein in a combination treatment with PR, RTI and/or NRTI.

The present invention also relates to a method of treating a symptom ofacquired immune deficiency syndrome in a human subject infected with HIVthat is, or has become, resistant to a non-antibody CD4 and/or CR (e.g.CXCR4) antagonist, comprising administering to the human subject apolypeptide of the invention, in an amount effective to treat thesymptom of acquired immune deficiency syndrome in the human subject.

In one aspect, the disclosure provides a method for suppressinginfection of a cell expressing CD4 and/or a CR, such as e.g. CXCR4, by avirus, the method comprising contacting the cell expressing CD4 and/or aCR, such as e.g. CXCR4 with any of the polypeptide constructs of theinvention to suppress infection of the cell by the virus. In someembodiments, the method allows for the suppression of the infection of acell in vitro, i.e., wherein the cell is not in a subject. In someembodiments, the method allows for the suppression of the infection of acell in vivo, i.e., wherein the cell is in a subject.

In an embodiment, the present invention provides a method for inhibitingbinding of an HIV to CR, the method comprising contacting CR with apolypeptide of the invention to inhibit binding of the virus to CR,wherein contacting CR with the polypeptide inhibits binding of HIV toCR, and wherein contacting CR with the polypeptide does not displace anatural ligand from CR.

In an embodiment, the present invention provides a method for decreasingdisplacement of a natural ligand bound from CR by an HIV, the methodcomprising contacting CR with a polypeptide of the invention, whereinthe contacting decreases the displacement of the natural ligand from CRby the HIV.

In an embodiment, the present invention provides a method forsuppressing infection of a cell expressing CR by a virus, the methodcomprising contacting the cell expressing CR with a polypeptide of theinvention to suppress infection of the cell by the virus, preferably,wherein contacting CR with the polypeptide does not displace a naturalligand from said CR.

In an embodiment, the present invention provides a method of inhibitingin a human subject the onset or progression of an HIV-associateddisorder, the inhibition of which is effected by inhibiting fusion of anHIV having resistance to (i) one or more HIV protease inhibitors, (ii)one or more HIV reverse transcriptase inhibitors, (iii) one or more HIVprotease inhibitors and one or more HIV reverse transcriptaseinhibitors, or (iv) one ISV of a polypeptide of the invention, to CXCR4CD4⁺ target cells in the subject, comprising administering to thesubject at a predefined interval effective fusion-inhibitory dose of apolypeptide of the invention, preferably wherein each administration ofthe polypeptide delivers to the subject from 0.1 mg per kg to 25 mg perkg of the subject's body weight, so as to thereby inhibit the onset orprogression of the HIV-associated disorder in the subject.

In one aspect, the disclosure provides a method for preventing HIVinfection in a subject, the method comprising administering to thesubject an immunoglobulin single variable domain or a polypeptideconstruct comprising one or more immunoglobulin single variable domainsto prevent HIV infection in the subject. In some embodiments, theadministered immunoglobulin single variable domain or polypeptideconstructs thereof inhibits binding of HIV to CD4 and/or a CR, such ase.g. CXCR4 but does not displace a natural ligand from said CR such ase.g. CXCR4. In some embodiments, the administered immunoglobulin singlevariable domain or polypeptides construct thereof have only minimalundesirable side effects.

The present invention relates to a method for preventing HIV infectionin a subject, the method comprising administering to the subject atherapeutically effective amount of a polypeptide as described herein toprevent infection of the subject by HIV.

In one aspect, the disclosure provides methods of preventing HIVinfection in a subject. In some embodiments, preventing HIV infection isachieved by precluding HIV from entering and/or accumulating in CD4⁺T-cells in the subject. Thus, in some embodiments, infection by HIV isprevented even after a subject has been exposed to an HIV, and may haveone or more signs of having been exposed to HIV, by preventing HIV fromentering and/or accumulating in the CD4⁺ T-cells in the subject.

Preventing HIV infection refers both to complete and partial prevention(e.g., a percentage reduction, for example about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or higher or lower or intermediate percentages of gettinginfected by HIV). For instance, a subject may have a 50% chance ofgetting infected by HIV upon exposure to the HIV through a specificroute (e.g., intravenous injection), but administration of theimmunoglobulin single variable domains disclosed herein or polypeptideconstructs thereof results in only a 10% chance of getting infected uponexposure (thus resulting in an 80% reduction in the chance of gettinginfected).

Prevention of infection can be determined using established simianmodels of HIV and SIV infection.

For instance, a group of animals (e.g., monkeys) can be administered theimmunoglobulin single variable domains or polypeptides constructsthereof and subsequently be exposed to HIV/SIV, while a control group,which is was also exposed to HIV/SIV, was not administered theimmunoglobulin single variable domains or polypeptides constructsthereof. If the incidence of HIV/SIV infection in the group to which theimmunoglobulin single variable domains or polypeptides constructsthereof have been administered is lower than in the control group, thenthe immunoglobulin single variable domains and constructs thereof areeffective in preventing infection by HIV.

In an embodiment, the present invention provides a method of reducingthe likelihood of a human subject's contracting infection by an HIVhaving resistance to (i) one or more HIV protease inhibitors, (ii) oneor more HIV reverse transcriptase inhibitors, (iii) one or more HIVprotease inhibitors and one or more HIV reverse transcriptaseinhibitors, or (iv) one ISV of a polypeptide of the invention, whichcomprises administering to the subject at a predefined schedule thepolypeptide of the invention, preferably wherein each administration ofthe polypeptide delivers to the subject from 0.1 mg per kg to 25 mg perkg of the subject's body weight, so as to thereby reduce the likelihoodof the subject's contracting an infection by a resistant HIV.

In some embodiments, the methods of treatment comprise administering oneor more of the immunoglobulin single variable domains and thepolypeptide constructs comprising immunoglobulin single variable domainsdescribed herein and one or more known or putative anti-viral compoundsor compounds displaying anti-viral activity. Known or putativeanti-viral compounds are compounds that suppress or inhibit viralinfection, viral replication and/or the development of diseaseassociated with viral infection. In some embodiments, the known orputative anti-viral compound is a known or putative anti-HIV compound.

Anti-viral drugs can be classified as targeting one of the life cyclestages of the virus. One category of anti-viral drugs are based oninterfering with viral entry. As described herein, a virus binds to aspecific receptor to infiltrate a target cell. Viral entry can besuppressed by blocking off the viral entry way. Anti-viral drugs thathave this mode of action are anti-receptor antibodies, natural ligandsof the receptor and small molecules that can bind to the receptor. Asecond category of anti-viral drugs are compounds that suppress viralsynthesis. Anti-viral drugs that have this mode of action are nucleosideanalogues that are similar to the DNA and RNA building blocks butdeactivate the protein machinery (e.g., reverse transcriptase or DNApolymerase) used to replicate the virus. Other drugs are targeted atblocking the transcription factors of viral DNA, ribozymes, which caninterfere with the production of viral DNA. Other drugs target viral RNAfor destruction, including siRNAs and antisense nucleic acids againstviral nucleic acid sequences. Yet another class of anti-viral drugsrelates to drugs that can interfere with the function of virus specificproteins. This class includes the HIV protease inhibitors. Anti-viraldrugs also include drugs directed at the release stage of the virus.This category of drugs includes compounds that interfere with theproteins necessary to build the viral particles. Another class ofanti-viral drugs are drugs that stimulate the immune system in targetingviral infection. Drugs that fall in this class are interferons, whichinhibit viral synthesis in infected cells and antibodies that can targetan infected cell for destruction by the immune system. Other anti-viralagents are described in U.S. Pat. Nos. 6,130,326, and 6,440,985, andpublished US patent application 2002/0095033. Accordingly, it should beappreciated that compounds identified herein have anti-viral activityand may act through any anti-viral mechanism described above. In someembodiments, compounds identified herein inhibit or suppress viralreplication (e.g., viral DNA replication).

In some embodiments, the anti-viral compounds are anti-viral compoundsthat are anti-HIV compounds. In some embodiments, the anti-viralcompounds are used in anti-HIV therapy, such as for instance, theanti-viral compounds described in Tables 1, 4 and 5 of WO2009/014638. Insome embodiments, the anti-HIV compound is an HIV protease inhibitor orHIV reverse transcriptase inhibitor.

The anti-viral activity of a compound may be assayed in an in vitro cellbased assay. Anti-viral activity may result from i) the interaction of acompound with the virus to prevent infection of a cell or to preventreplication, development, and/or proliferation of the virus afterinfection, ii) the effect of a compound on a cell to prevent infectionby the virus or to prevent replication, development, and/orproliferation of the virus after infection, or iii) any other mechanism,or any combination thereof.

Regardless of the mode of action, a composition may have anti-viralactivity if it reduces the percentage or number of infected cells in acell-based assay. In some embodiments, a compound (or a combination oftwo or more compounds) has anti-viral activity when it reduces thepercentage or number of infected cells by at least 20%, at least 30%, atleast 40%, at least 50%, or more (e.g., in a cell-based assay). In someembodiments, a compound has anti-viral activity when it reduces theamount of viral nucleic acids within a cell. In certain embodiments, acompound inhibits the replication of viral nucleic acids within a cell(e.g., a compound reduces the amount of viral replication by about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or higher or lower or intermediatepercentages of reduction). It should be appreciated that a reduction inviral replication may be measured using a cellular assay and measuringthe amount of viral DNA or the rate of viral DNA replication over time(or any other measure of viral replication) in the presence of acompound and comparing it to the viral replication in the absence of thecompound or in the presence of a control compound.

In some embodiments, the methods of treatment and/or prevention compriseadministering one or more of the immunoglobulin single variable domainsand the polypeptide constructs comprising immunoglobulin single variabledomains described herein and administering a vaccine against a DNAvirus. A vaccine is defined as a pharmaceutical composition that whenadministered to a subject in an effective amount stimulates theproduction of protective antibody or protective T-cell response. In someembodiments, the vaccine is protein vaccine comprising one or morepolypeptide sequences encoded by a DNA virus sequence. In someembodiments, the vaccine is a nucleic acid vaccine comprising DNA viralnucleic acids. Administration regimes for vaccines are known to a personof ordinary skill in the art. In some embodiments, ranges of amounts ofpolypeptide vaccines for prophylaxis of DNA viral infection are from0.01 to 100 microgram/dose, for example 0.1 to 50 microgram/dose.Several doses may be needed per subject in order to achieve a sufficientimmune response and subsequent protection against DNA viral infection(e.g., “immunizing” a subject). The term “immunizing” refers to theability of a substance to cause a humoral and/or cellular response in asubject, whether alone or when linked to a carrier, in the presence orabsence of an adjuvant, and also refers to an immune response thatblocks the infectivity, either partially or fully, of an infectiousagent.

In some embodiments, the methods of treatment or prevention compriseadministering one or more of the immunoglobulin single variable domainsand the polypeptide constructs comprising immunoglobulin single variabledomains described herein and administering a compound or therapy thatreduces unwanted side effects of HIV-therapy. Examples of such compoundsinclude anti-nausea, appetite enhancers and anti-depressants.

In one aspect, the disclosure provides methods for the administration oftherapeutically effective amounts of immunoglobulin single variabledomains and polypeptide constructs comprising one or more immunoglobulinsingle variable domains. A therapeutically effective amount of an ISV orpolypeptide is a dosage of the immunoglobulin single variable domain orpolypeptide to provide a medically desirable result (e.g., lowering ofthe HIV-titer). The effective amount will vary with the particularcondition being treated, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent therapy (if any), the specificroute of administration and like factors within the knowledge andexpertise of the health practitioner. For example, a therapeuticallyeffective amount for treating or preventing a disease or condition(e.g., suffering from infection by HIV) would be an amount sufficient todecrease the progression of, or inhibit the disease or condition, or itssymptoms. Similarly, a therapeutically effective amount for lowering theHIV titer in a subject would be an amount sufficient to lower the HIVtiter in a subject. It should be appreciated that non-immunoglobulinsingle variable domains therapies can be administered in therapeuticallyeffective amounts as well.

In one aspect, the disclosure provides methods for the administration ofimmunoglobulin single variable domain and polypeptide constructs thereofcomprising one or more immunoglobulin single variable domains. In someembodiments, the immunoglobulin single variable domain or polypeptide isadministered as a pharmaceutical composition. The pharmaceuticalcomposition, in addition to the immunoglobulin single variable domainsand polypeptide constructs thereof includes apharmaceutically-acceptable carrier.

As described in detail, the pharmaceutical compositions of thedisclosure may be specially formulated for administration in solid orliquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Formulations of the disclosure include those suitable for oral, nasal,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,immunoglobulin single variable domain or polypeptide constructs thereof)which can be combined with a carrier material to produce a single dosageform will vary depending upon the host being treated, and the particularmode of administration. The amount of active ingredient that can becombined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, this amount will range from about 1% to about 99% ofactive ingredient, preferably from about 5% to about 70%, mostpreferably from about 10% to about 30%.

In certain embodiments, a formulation comprises an excipient selectedfrom the group consisting of cyclodextrins, liposomes, micelle formingagents, e.g., bile acids, and polymeric carriers, e.g., polyesters andpolyanhydrides. In certain embodiments, an aforementioned formulationrenders orally bioavailable an immunoglobulin single variable domain orpolypeptide construct.

Methods of preparing these formulations or compositions include the stepof bringing into association an immunoglobulin single variable domain orpolypeptide construct with the carrier and, optionally, one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association an immunoglobulinsingle variable domain or polypeptide construct with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of an immunoglobulin single variable domain orpolypeptide construct as an active ingredient. An immunoglobulin singlevariable domain or polypeptide construct invention may also beadministered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glycerol; disintegrating agents, such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; solution retarding agents, such asparaffin; absorption accelerators, such as quaternary ammoniumcompounds; wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; absorbents, such as kaolin andbentonite clay; lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxy-propylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent.

Molded tablets may be made in a suitable machine in which a mixture ofthe powdered compound is moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules, pills and granules, mayoptionally be scored or prepared with coatings and shells, such asenteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing an immunoglobulin single variable domain or polypeptideconstruct with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations suitable for vaginal administration also include pessaries,tampons, creams, gels, pastes, foams or spray formulations containingsuch carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of animmunoglobulin single variable domain or polypeptide construct includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically-acceptable carrier, and with anypreservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an immunoglobulin single variable domain or polypeptideconstruct to the body. Dissolving or dispersing the compound in theproper medium can make such dosage forms. Absorption enhancers can alsobe used to increase the flux of the compound across the skin. Eitherproviding a rate controlling membrane or dispersing the compound in apolymer matrix or gel can control the rate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more an immunoglobulin single variable domains orpolypeptide constructs in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers, which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly-(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, and copending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

The Figures and the Experimental Part/Examples are only given to furtherillustrate the invention and should not be interpreted or construed aslimiting the scope of the invention and/or of the appended claims in anyway, unless explicitly indicated otherwise herein.

TABLE A-2CDR's and framework sequences of Nanobodies against human CXCR4 (“ID”represents SEQ ID NO:”) ID Clone ID Framework 1 ID CDR 1 ID Framework 2ID CDR 2  4 238 D4 21 EVQLMESGGGLVQAGGSL 34 NYAMG 41 WFRRAPGKEREFVA 48AITRSGVRSGVSAIYGDS RLSCAASGRTFN VKG  5 281A5 22 EVQLVESGGGLVQAGGSL 34NYAMG 41 WFRRAPGKEREFVA 48 AITRSGVRSGVSAIYGDS RLSCAASGRTFN VKD  6 281E1023 EVQLVESGGGLVQAGGSL 34 NYAMG 41 WFRRAPGKEREFVA 48 AITRSGVRSGVSAIYGDSRLSCKASGGTFN VKD  7 281D4 24 EVQLVESGGGLVQAGGSL 34 NYAMG 41WFRRAPGKEREFVA 49 AISRSGVRTGVSALYGDS RLSCAASGGTFN VKD  8 281A6 25EVQLVESGGGLVQTGGSL 34 NYAMG 41 WFRRAPGKEREFVA 49 AISRSGVRTGVSALYGDSRLSCAASGGTFN VKD  9 281F12 26 EVQLVESGGGLVQAGDSL 35 RYAMG 42WFRQAPGKEREFVA 50 AIGWGPSKTNYADSVKG RLSCAASGRAFS 10 283B6 27EVQLVASGGGLVQAGGSL 36 VATLG 43 WYRQAPGQQRALVA 51 DISSGGSTNYADSVRGRLSCAVSGTTFS 11 283E2 28 EVQLVESGGGLVQAGGSL 36 VATLG 43 WYRQAPGQQRALVA51 DISSGGSTNYADSVRG RLSCAVSGTTFS 12 283F1 29 EVQLVESGGGLVQAGGSL 37 SIAMA44 WYRQAPGKQRNLVA 52 SISSGGRINYADSRKG RLSCVASVNIFG 13 15F5 30EVQLVESGGGLVRAGDSL 35 RYAMG 45 WFRQALGKERELVA 53 AIGWSPTHTYYADSVKGRLSCAASGRAFS 14 15G11 31 EVQLVESGGGLVQAGDSL 38 SYAMA 46 WFRQAPGKEREFVG54 TISRTNSRTKYADFVEG RVSCAASGRTS 15 15A1 32 EVQLVESGGGLVQAGGSL 39 SRAAMG46 WFRQAPGKEREFVG 55 CALSSAGSALTADSVKG RLSCAASGRTF 16 10C03 33EVQLVESGGGLVQPGGSL 40 STSTMG 47 WYSQAPGKQRELVA 56 DITFLGSAKYADSVKGRLSCAASGTIF ID ID Framework 3 ID CDR 3 ID Framework 4  4 57RFTISRDNAKNTLYLQMNSLKPEDTAVYT 67 SAIGSGALRRFEYD 76 SGQGTQVTVSS CAA Y  557 RFTISRDNAKNTLYLQMNSLKPEDTAVYT 67 SAIGSGALRRFEYD 76 SGQGTQVTVSS CAA Y 6 58 RFTISRDNVKNTLYLQMNTLKPEDTAVYT 67 SAIGSGALRRFEYD 76 SGQGTQVTVSS CAAY  7 59 RFTISRDNAKNTLYLQMNKMKPEDTAVYT 68 SAIGSGALRRFEYD 76 SGQGTQVTVSSCAA S  8 59 RFTISRDNAKNTLYLQMNKMKPEDTAVYT 68 SAIGSGALRRFEYD 76SGQGTQVTVSS CAA S  9 60 RFTISRDNAKNTVYLQMNTLKPEDTAVYS 69 KFVNTDSTWSRSEM77 WGQGTQVTVSS CAA YTY 10 61 RFTISRDNAKNLAYLQMNSLKPEDTAVYY 70RTSGWRTRSNY 77 WGQGTQVTVSS CNA 11 61 RFTISRDNAKNLAYLQMNSLKPEDTAVYY 70RTSGWRTRSNY 77 WGQGTQVTVSS CNA 12 62 RFTISRDNTKNTVHLQMNSLEPEDTAVYY 71GRIGQRTLTFTPDY 77 WGQGTQVTVSS CAA 13 63 RFTMSRDNGKNTVFLQMNSLNPEDTAVYY 72KYSSRDAAYRSDYD 77 WGQGTQVTVSS CAA YNY 14 64RFTISRDNAKSTLSLQMTSLKPEDTAVYY 73 KWTGNSYHDYTWSK 77 WGQGTQVTVSS CAAVDEYNV 15 65 RFTISRDNAKNMVYLQMNNLKPEDTAVYS 74 GGYCTRAGVYPY 77WGQGTQVTVSS CVA 16 66 RFTISRDKIKNTVYLQMNSLKPEDTAAYY 75 RQSTFRGVHYNY 77WGQGTQVTVSS CNACDR's and framework sequences of Nanobodies against human CD4 (“ID”represents SEQ ID NO:”) ID Clone ID FR1 ID CDR1 ID FR2 ID CDR2 17 01B678 EVQLVESGGGLVQSGGSL 82 GYWMY 86 WVRQAPGKGLEWVS 88 AISPGGGSTYYPDSVKGRLSCAASGFTFS 18 01E2 79 EVQLVESGGGLVQAGGSL 83 SYSMG 42 WFRQAPGKEREFVA 89AISWSGDETSYADSVKG RLSCAASGRTSA 19 01H12 80 EVQLVESGGGLVQAGGSL 84 FNAMG87 WYRQAPGKQREWVT 90 TIARAGATKYADSVKG KLSCAASRSILD 20 03F11 81EVQLVESGGGSVQPGGSL 85 VMG 42 WFRQAPGKEREFVA 91 AVRWSSTGIYYTQYADSVTLSCGTSGRTFN KS ID ID FR3 ID CDR3 ID FR4 17 92RFTISRDNAKNTLYLQMNSLKPEDTALYY 96 SLTATHTYEYDY  77 WGQGTQVTVSS CAS 18 93RFTIARGNAKNTVYLQMNSLKSEDTAIYY 97 DRWWRPAGLQWDY  77 WGQGTQVTVSS CAG 19 94RFSISRDNAKNTVYLQMSSLKPEDTATYY 98 RVFDLPNDY  77 WGQGTQVTVSS CNA 20 95RFTISRDNAKNTVYLEMNSLKPEDTAVYY 99 DTYNSNPARWDGYD 100 RGQGTQVTVSS CAA F

EXPERIMENTAL SECTION Example 1: Introduction

HIV diversity is so extensive, especially in the envelope gp120, thatdesigning an HIV vaccine capable of eliciting broadly cross-reactiveneutralizing antibodies is an extraordinarily difficult challenge.

The virus replicates rapidly and has a high mutation rate creatinghighly diverse ‘quasi species’. These quasi species are fertilesubstrates for Darwinian selective pressures favoring the best-adapted,most ‘fit’ genetic variants. Efforts to develop effective treatments andvaccines must overcome the complex evolutionary dynamics in HIV-infectedindividuals and within affected populations.

As HIV spreads from individual to individual, genetically diverseviruses confront the most highly polymorphic gene family in humans—thatencoding the human leukocyte antigen (HLA) class I and II proteins.These proteins determine which specific peptide sequences (epitopes) arepresented to and recognized by host CD8⁺ and CD4⁺ T-cells, respectively.In the confrontation between genetically diverse HIV variants andgenetically diverse human hosts, viral variants can be selected thatharbor mutations in specific viral epitopes that escape recognition byhost immune effector cells, resulting in resistant HIV.

However, although the viral diversity is extremely high, most HIVgroups, strains, clades and subtypes are in need of the cellularreceptor CD4 as well as the co-receptors CXCR4 (X4), CCR5 (R5) or bothCCR5/CXCR4 (dual tropic R5/X4), to enter and infect mainly the CD4⁺target immune T-cells (see FIG. 1).

The present inventors hypothesized that the simultaneous blockade ofboth the receptor and a co-receptor could not only prevent HIV entry,but also defer if not preclude resistance. Surprisingly, the use ofbispecific CXCR4-CD4 polypeptides outperformed the combination of theindividual blockers and could prevent HIV entry and overcome resistance.

Example 2: Identification and Characteristics of Monovalent CD4Nanobodies Example 2.1: Selection of CD4 Nanobody Candidates

A panel of CD4 Nanobodies was previously identified from immunelibraries with human peripheral blood lymphocytes. Llama 58, 59 and 60were immunized according to standard protocols with 6 boosts at two weekintervals, each of them with approximately 1×10⁸ human peripheral bloodlymphocytes (hPBLs). Blood was collected 4 and 9 days after the finalboost. In addition, approximately 1 g of lymph node biopsies werecollected from animals 4 days after final boost. Peripheral bloodmononuclear cells were prepared from blood samples using Ficoll-Hypaqueaccording to the manufacturer's instructions. Next, total RNA wasextracted from these cells and lymph node tissue, if available, and usedas starting material for RT-PCR to amplify Nanobody encoding genefragments. These fragments were cloned into phagemid vector pAX50. Phagewas prepared according to standard methods (see for example the priorart and applications filed by applicant cited herein).

Selections of phage displaying CD4 binding Nanobodies Phage libraries58, 59 and 60 were used for selections on recombinant human CD4(ImmunoDiagnostics, Inc., cat#7001, lot #5S30/1.5). Recombinant humanCD4 was immobilized directly on Maxisorp 96 well microtiter plates(Nunc) at 10 μg/ml, 0.1 μg/ml and 0 μg/ml (control). Followingincubation with the phage libraries and extensive washing, bound phagewas eluted with 100 mM triethylamine (TEA). The eluted phage wereamplified and applied in a similar second round of selection. Afterelution with TEA the second round obtained phage were again amplifiedand applied into a similar third round selection in which followingincubation with the phage and extensive washing, bound phage wasa-specifically eluted with TEA, or specifically with 250 nM of gp120HIV-1 IIIB (Immunodiagnostic). Individual colonies obtained from theeluted phage pools were grown and i) induced for new phage productionand ii) induced with IPTG for Nanobody expression and extraction(periplasmic extracts) according to standard methods (see for examplethe prior art and applications filed by applicant cited herein).

2.2 Screening for CD4 Binding Nanobodies

In order to determine binding specificity to CD4, the selected cloneswere tested in an ELISA binding assay setup, using the monoclonal phagepools. Shortly, 1 μg/ml receptor recombinant human CD4(ImmunoDiagnostics Inc., cat#7001) was immobilized on Maxisorp ELISAplates (Nunc) and free binding sites were blocked using 4% Marvelskimmed milk in PBS. Next, 15 μl of supernatant from the monoclonalphage inductions of the different clones in 100 μl 1% Marvel PBS wereallowed to bind to the immobilized antigen. After incubation and a washstep, phage binding was revealed using a HRP-conjugatedmonoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificitywas determined based on OD values compared to controls having receivedno phage or an irrelevant phage. FIG. 2 shows binding of 4 clones torecombinant human CD4 in phage ELISA. The sequences are depicted inTable 2.2

TABLE 2.2 CD4 Nanobodies >01B6 (SEQ ID NO: 17)EVQLVESGGGLVQSGGSLRLSCAASGFTFSGYWMYWVRQAPGKGLEWVSAISPGGGSTYYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCASSLTATHTYEYDYWGQGTQVTVSS >01E2 (SEQ ID NO: 18)EVQLVESGGGLVQAGGSLRLSCAASGRTSASYSMGWFRQAPGKEREFVAAISWSGDETSYADSVKGRFTIARGNAKNTVYLQMNSLKSEDTAIYYCAGDRWWRPAGLQWDYWGQGTQVTVSS >01H12 (SEQ ID NO: 19)EVQLVESGGGLVQAGGSLKLSCAASRSILDFNAMGWYRQAPGKQREWVTTIARAGATKYADSVKGRFSISRDNAKNTVYLQMSSLKPEDTATYYCNARVFDLPNDYWGQGTQVTVSS >03F11 (SEQ ID NO: 20)EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTQVTVSS

CD4 binding Nanobodies were subjected to sequence analysis, and uniqueclones were recloned into an E. coli expression vector, allowing furthercharacterisation as purified Nanobodies. Monovalent CD4 Nanobodies wereproduced as C-terminal linked myc, His6-tagged proteins in expressionvector pAX50.

To check whether the selected Nanobodies recognize cell-surfaceexpressed CD4, a Flow cytometry experiment was performed where thepurified CD4 Nanobodies were tested for specific binding to Jurkat andTHP-1 cells expressing human CD4. Murine Ba/F3 myeloid cells were usedas negative control cells. Purified Nanobodies (100 nM) were allowed tobind to 10⁵ cells for 30 minutes at 4° C. in a final volume of 100 μl of10% FBS (Invitrogen, Cat 10270-106) in PBS (Invitrogen #14190). BoundNanobodies were detected with mouse anti-myc antibody (Serotec,Cat#MCA2200) followed by goat anti-mouse-PE antibody (Jackson#115-115-164). Dead cells percentage population was determined bystaining the cells with TOPRO3 (Molecular probes T3605). Using a BD FACSArray Bioanalyzer system, a PE filter 585/42 and a Topro filter 661/16,twenty thousand events were acquired. TOPRO3+ cells were excluded, andthe mean channel fluorescence (MCF) was calculated. Expression of CD4 inJurkat and THP-1 cells was confirmed by using 10 μg/ml of an anti-CD4monoclonal antibody (Diaclone, clone B-A1 cat#854.030.000). Staining ofthe cells with anti-myc and/or the goat anti mouse-PE antibodiesantibody was performed as negative controls. The results obtained for 4different Nanobodies and control antibodies are shown in FIG. 2,confirming that all Nanobodies are binding to CD4 expressed on a cellsurface, with Nanobody 03F11 showing the highest binding signal on THP-1cells.

2.3 Screening for Nanobodies Blocking CD4-Gp120 Interaction

Besides its role on T-cells, CD4 also serves as primary receptor for HIVentry. Therefore purified CD4 Nanobodies were analysed for the capacityto block the interaction of CD4 with the viral gp120 protein. MonovalentHis-tagged Nanobodies were purified from periplasmic extracts byaffinity and desalting chromatography and used in an ELISA-basedcompetition setup. In short, 1 μg/ml of gp120 HIV-1 IIIB(Immunodiagnostic) was captured by 20 μg/ml of sheep anti-gp120 antibodyD7324 (Aalto Bio Reagents) previously coated in 96 well Maxisorpmicrotiter plates (Nunc) and blocked with 1% casein in PBS. In parallel,0.5 μg/ml of biotinylated CD4 was incubated with 500 nM of the differentpurified Nanobodies in 100 μl 0.1% Casein/PBS. After 1 hour, thebiotinylated CD4-Nanobody pre-mixes were incubated 1 hour with thecaptured gp120. Bound biotinylated CD4 was detected using HRP-conjugatedExtravidin (Sigma E2886). A blocking mouse anti-CD4 IgG2a antibody(Diaclone, clone B-A1 cat#854.030.000) was used as positive control.Blocking activity was determined as loss of O.D. signal, as compared towells where no Nanobody was added.

FIG. 2 panel C shows results of this blocking assay using a selection ofclones binding to human CD4.

The results indicate that Nanobodies 03F11 and 01B6 block the in vitrointeraction of CD4 with gp120.

2.4 Characterisation of CD4 Nanobody 3F11

Nanobody 03F11 (also designated as 3F11) was subsequently analysed fordose-dependent binding to primary human T cells, MOLM-13 and THP-1 cellsin FACS, using detection of the anti-flag-tag. The EC50 values resultsare depicted in Table 2.4. Also on primary cells, 03F11 showed strongbinding, with an EC₅₀ value of 0.76 nM (FIG. 2 Panel D). To confirm thespecificity of the anti-CD4 Nanobody, binding of 03F11 was also assessedto cytotoxic CD8⁺ T cells isolated from human PBMCs using the CD8 T CellIsolation Kit (Miltenyi Biotech, Cat. 130-096-495), resulting in 94%purity of CD8⁺ cells. No binding was observed to cytotoxic CD8⁺ T cellswith the anti-CD4 03F11 Nanobody (data not shown).

The results indicate that Nanobodies are capable of binding to thereceptor CD4 and preventing the interaction with the HIV-1 gp120binding.

TABLE 2.4 Characteristics of monovalent CD4 Nanobody 3F11. HIV-1 FACSbinding neutralization MOLM-13 THP-1 T-cells MT-4 + NL4.3 Nanobody IDEC₅₀ (nM) EC₅₀ (nM) EC₅₀ (nM) IC₅₀ (nM) CD4 3F11 0.6 1.0 0.76 34.7

Example 3 Identification and Selection of CXCR4 Nanobody Candidates

In the present example, the inventors set out to identify andcharacterize anti-CXCR4 Nanobodies which were able to act as functionalantagonists in HIV1 infectivity assays.

Preferably, these CXCR4 Nanobodies would bind to an epitope(s) thatinterferes with gp120 interaction but not with binding of the ligandCXCL12, because of which the Nanobodies would not interfere with naturalCXCR4 signal transduction.

3.1 Ligand Displacement of CXCR4 Nanobodies

To this end, CXCR4 Nanobodies were analysed for their binding toHEK293T-CXCR4 cells, and their ability to compete with the ligand CXCL12(or SDF-1a) for receptor binding in a radio-ligand displacement assay.The sequences of the respective CXCR4 Nanobodies are depicted in Table3.1A.

TABLE 3.1A CXCR4 Nanobodies >281A5 (SEQ ID NO: 5)EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS >281E10 (SEQ ID NO: 6)EVQLVESGGGLVQAGGSLRLSCKASGGTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNVKNTLYLQMNTLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS >281D4 (SEQ ID NO: 7)EVQLVESGGGLVQAGGSLRLSCAASGGTFNNYAMGWFRRAPGKEREFVAAISRSGVRTGVSALYGDSVKDRFTISRDNAKNTLYLQMNKMKPEDTAVYTCAASAIGSGALRRFEYDSSGQGTQVTVSS >281A6 (SEQ ID NO: 8)EVQLVESGGGLVQTGGSLRLSCAASGGTFNNYAMGWFRRAPGKEREFVAAISRSGVRTGVSALYGDSVKDRFTISRDNAKNTLYLQMNKMKPEDTAVYTCAASAIGSGALRRFEYDSSGQGTQVTVSS >281F12 (SEQ ID NO: 9)EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGWGPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSS >283B6 (SEQ ID NO: 10)EVQLVASGGGLVQAGGSLRLSCAVSGTTFSVATLGWYRQAPGQQRALVADISSGGSTNYADSVRGRFTISRDNAKNLAYLQMNSLKPEDTAVYYCNARTSGWRTRSNYWGQGTQVTVSS >283E2 (SEQ ID NO: 11)EVQLVESGGGLVQAGGSLRLSCAVSGTTFSVATLGWYRQAPGQQRALVADISSGGSTNYADSVRGRFTISRDNAKNLAYLQMNSLKPEDTAVYYCNARTSGWRTRSNYWGQGTQVTVSS >283F1 (SEQ ID NO: 12)EVQLVESGGGLVQAGGSLRLSCVASVNIFGSIAMAWYRQAPGKQRNLVASISSGGRINYADSRKGRFTISRDNTKNTVHLQMNSLEPEDTAVYYCAAGRIGQRTLTFTPDYWGQGTQVTVSS 238D4 (SEQ ID NO: 4)EVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS 15F5 (SEQ ID NO: 13)EVQLVESGGGLVRAGDSLRLSCAASGRAFSRYAMGWFRQALGKERELVAAIGWSPTHTYYADSVKGRFTMSRDNGKNTVFLQMNSLNPEDTAVYYCAAKYSSRDAAYRSDYDYNYWGQGTQVTVSS 15G11 (SEQ ID NO: 14)EVQLVESGGGLVQAGDSLRVSCAASGRTSSYAMAWFRQAPGKEREFVGTISRTNSRTKYADFVEGRFTISRDNAKSTLSLQMTSLKPEDTAVYYCAAKWTGNSYHDYTWSKVDEYNVWGQGTQVTVSS 15A1 (SEQ ID NO: 15)EVQLVESGGGLVQAGGSLRLSCAASGRTFSRAAMGWFRQAPGKEREFVGCALSSAGSALTADSVKGRFTISRDNAKNMVYLQMNNLKPEDTAVYSCVAGGYCTRAGVYPYWGQGTQVTVSS 10C3 (SEQ ID NO: 16)EVQLVESGGGLVQPGGSLRLSCAASGTIFSTSTMGWYSQAPGKQRELVADITFLGSAKYADSVKGRFTISRDKIKNTVYLQMNSLKPEDTAAYYCNARQSTFRGVHYNYWGQGTQVTVSS

In short, membrane extracts of HEK293 cells transiently transfected withCXCR4 were incubated with serial dilutions of purified Nanobodies and 75pM of [¹²⁵1]-CXCL12. Non-specific binding was determined in presence of100 nM cold SDF-1. The assay was performed three times, and averagepercentages of SDF-1 inhibition and Ki values were calculated (Table3.1B).

TABLE 3.1B Ligand displacement affinity of monovalent CXCR4 Nanobodies.[¹²⁵I] SDF-1 displacement on CXCR4-Hek ID pKi +/SEM (n = 3 281F12 7.57+/− 0.30 281D4 8.27 +/− 0.05 281A6 8.63 +/− 0.02 281E10 9.21 +/− 0.12283B6 8.78 +/− 0.24 283E2 8.29 +/− 0.11 281A5 8.12 +/− 0.08 283F1 7.93+/− 0.41

In FIG. 2.1, is shown that 281F12 had a moderate potency, with a Ki of27 nM, and only partial efficacy, while other CXCR4 Nanobodies showedfull efficacy in displacing the binding of ligand to the CXCR4 receptor.

This would indicate that Nanobody 281F12 does not or only minimallyimpairs natural CXCR4 signal transduction.

3.2 Inhibition of HIV-1 Replication by Nanobodies

To determine if monovalent CXCR4 Nanobodies as well as the monovalentCD4 3F11 Nanobody are capable of blocking the replication of theCXCR4-using HIV1 strains, HIV-1 infection assays were performed withboth CXCR4 and CCR5 specific HIV clones.

The NL4.3, the CCR5-using (R5) HIV-1 strain BaL, the dual-tropic (R5/X4)HIV-1 strain HE and the dual-tropic (R5/X4) HIV-2 ROD strain wereinvestigated on human MT-4 cells, that endogenously express CD4 andCXCR4, but not CCR5. Activity (IC₅₀) and toxicity (CC₅₀) were determinedusing microscopic evaluation and MTS viability staining method. TheCXCR4-using (X4) HIV-1 clone NL4.3 was obtained from the NationalInstitutes of Health NIAID AIDS Reagent program (Bethesda, Md.), theCCR5-using (R5) HIV-1 strain BaL was obtained from the Medical ResearchCouncil AIDS reagent project (Herts, UK). The dual-tropic (R5/X4) HIV-1HE strain was initially isolated from a patient at the UniversityHospital in Leuven. In all experiments AMD3100, a specific CXCR4antagonist, and maraviroc, a specific CCR5 antagonist, were used ascontrols. The MT-4 cells were seeded out in 96-well plates. Nanobodieswere added at different concentrations together with HIV-1 and theplates were maintained at 37° C. in 10% CO₂. Cytopathic effect inducedby the virus was monitored by daily microscopic evaluation of thevirus-infected cell cultures. At day 4-5 after infection, when strongcytopathic effect was observed in the positive control (i.e., untreatedHIV-infected cells), the cell viability was assessed via the in situreduction of the tetrazolium compound MTS, using the CellTiter 96®AQ_(ueous) One Solution Cell Proliferation Assay (Promega, Madison,Wis.). The absorbance was measured spectrophotometrically at 490 nm witha 96-well plate reader (Molecular Devices, Sunnyvale, Calif.) andcompared with four cell control replicates (cells without virus anddrugs) and four virus control wells (virus-infected cells withoutdrugs). The IC₅₀, i.e., the drug concentration that inhibits HIV-inducedcell death by 50%, was calculated for each polypeptide from thedose-response curve. The CC₅₀ or 50% cytotoxic concentration of each ofthe polypeptides was determined from the reduction of viability ofuninfected cells exposed to the agents.

The respective IC₅₀ values for the CXCR4 Nanobodies on MT-4 cells aredepicted in Table 3.2. The IC₅₀ values for the CD4 Nanobodies aredepicted in Table 2.4. Nanobody 03F11 directed against CD4 inhibits X4HIV-1 NL4.3 replication with an IC₅₀ of 0.52 μg/ml in MT-4 cells,corresponding to 34.7 nM.

The anti-CXCR4-directed Nanobody 281F12 had a comparable potency andinhibited HIV-1 NL4.3 replication with an average IC₅₀ of 0.34 μg/ml,corresponding to 22.7 nM. The same set of CXCR4 Nanobodies were alsoevaluated on human PBMCs (endogenously expressing subpopulations of CD4,CXCR4 and CCR5) against HIV-1 X4 NL4.3 strain, HIV-1 X4 UG270 clinicalisolate clade D, X4 HIV-1 CI#17 clinical isolate clade B, X4 HIV-1 CM237clinical isolate clade B and against HIV-1 R5 BaL strain. Again, AMD3100and AMD14031 (maraviroc) were used as controls in all experiments.Peripheral blood mononuclear cells (PBMCs) from healthy donors wereisolated by density centrifugation (Lymphoprep; Nycomed Pharma, ASDiagnostics, Oslo, Norway) and stimulated with phytohemagglutin for 3days. The activated cells were washed with PBS and viral infections wereperformed as described previously (Schols et al. J Exp Med 1997;186:1383-1388). PHA-stimulated blasts were seeded at 0.5×10⁶ cells perwell into a 48-well plate (Costar; Elscolab, Kruibeke, Belgium)containing varying concentrations of compound in medium containing IL-2.The virus stocks were added at a final dose of 100 TCID50 of HIV-1 orHIV-2. At 8-10 days after the start of the infection, viral p24 Ag wasdetected in the culture supernatant by an enzyme-linked immunosorbentassay (Perkin Elmer, Brussels, Belgium). For HIV-2 p27 Ag detection, theINNOTEST from Innogenetics (Temse, Belgium) was used.

Table 3.2 summarizes the results of the HIV neutralization on 4 X4 HIV-1strains by CXCR4 Nanobodies.

The results indicate that CXCR4 Nanobodies showed consistentneutralization capacity on different clinical isolates that aredependent on CXCR4, whereas none of these was able to block infection ofthe BaL strain (IC₅₀>1000 ng/mL, data not shown). Of the tested panel,281E10 and 283F1 were very potent antagonists on all X4 strains, whereas281F12 was the least potent Nanobody, with IC₅₀ ranging between 9-16 nM.The potency in HIV1 neutralisation is in the same affinity range as theligand displacement Ki of 26 nM.

The inhibition of HIV-1 replication (combined with its poor liganddisplacement capacity) makes CXCR4 Nanobody 281F12 a suitable initialcandidate for use in formatting into bispecific constructs with CD4Nanobody 3F11.

TABLE 3.2 CXCR4 Nanobodies were evaluated for blocking the infectivityof distinct clinical HIV1 isolates in PBMC, i.e. HIV-1 X4 NL4.3 strain,HIV-1 X4 UG270 clinical isolate (clade D), HIV-1 CI#17 clinical isolate(clade B), HIV-1 CM237 clinical isolate (clade B) and against HIV-1 R5BaL strain. The Nanobodies are ranked according to potency. As controlcompounds AMD3100 (specific CXCR4 antagonist) and maraviroc (specificCCR5 antagonist) were included. M, HIV1 strain Average NL4.3 UG270 CI#17CM237 compound 2 donors X4 Clade D Clade B Clade B 283F1 IC₅₀ 7.6E−101.0E−09 2.0E−09 1.2E−09 IC₉₀ 3.6E−09 8.1E−09 9.1E−09 5.1E−09 281E10 IC₅₀1.2E−09 1.8E−09 1.3E−09 6.7E−10 IC₉₀ 3.5E−09 7.7E−09 8.1E−09 4.0E−09283B6 IC₅₀ 1.6E−09 1.9E−09 2.8E−09 2.0E−09 IC₉₀ 7.3E−09 6.4E−09 9.9E−097.3E−09 283E2 IC₅₀ 4.1E−09 1.3E−09 3.7E−09 3.3E−09 IC₉₀ 1.2E−08 1.4E−081.7E−08 1.7E−08 281D4 IC₅₀ 1.2E−09 7.8E−09 1.5E−08 3.3E−09 IC₉₀ 3.8E−092.7E−08 3.3E−08 1.0E−08 281A6 IC₅₀ 2.5E−09 1.1E−08 2.0E−08 9.9E−09 IC₉₀9.1E−09 2.7E−08 6.1E−08 2.7E−08 281A5 IC₅₀ 2.3E−08 4.0E−09 1.2E−081.8E−08 IC₉₀ 5.95E−08  7.33E−09  >5.7E−08  4.9E−08 281F12 IC₅₀ 1.4E−081.2E−08 8.9E−09 1.6E−08 IC₉₀ 5.7E−08 2.4E−09 4.0E−08 4.9E−08 irrelevantIC₅₀ >5.7E−08  >5.7E−08  >5.7E−08  >5.7E−08 IC₉₀ >5.7E−08  >5.7E−08  >5.7E−08  >5.7E−08  AMD3100 IC₅₀ 1.3E−081.6E−08 2.2E−08 1.0E−08 IC₉₀ 4.2E−08 4.1E−08 4.7E−08 2.4E−08

Example 4: Combination Studies with Monovalent CXCR4 and CD4 Nanobodies

To test the combinatorial effects of CXCR4 and CD4 Nanobodies on HIV-1activity, each of the selected Nanobodies was tested alone or incombination with another anti-HIV compound.

The anti-HIV compounds used were (1) AMD3100 (plerixafor, trade nameMozobil, Genzyme), which is a specific CXCR4 antagonist; (2) T-20(enfuvirtide, trade name FUZEON®, Roche), which is a gp41-mimickingpeptide acting as a HIV fusion inhibitor; (3) CADA(cyclotriaza-disulfonamide), which is a CD4 down-modulating compoundacting as a specific CD4-targeted HIV entry inhibitor; (4) RPA-T4(anti-CD4 mouse mAb), which binds to the D1 domain of CD4 and can blockHIV gp120 binding and inhibit syncytia formation.

The anti-HIV-1 EC₅₀ and EC₉₅ before and after combination weredetermined by measuring the cytopathic effect of NL4.3 in MT-4 cells,using the MTS viability staining method described above. Combinationindices (CI) were calculated using CalcuSyn software (Biosoft,Cambridge, UK) based on the median effect principle of Chou and Talalay(Chou and Talalay, 1984). The derived combination index equation for twodrugs is:

$\begin{matrix}{{CI} = {\frac{(D)_{1}}{\left( D_{x} \right)_{1}} + \frac{(D)_{2}}{\left( D_{x} \right)_{2}}}} \\{= {\frac{(D)_{1}}{{\left( D_{m} \right)_{1}\left\lbrack {f_{a}/\left( {1 - f_{a}} \right)} \right\rbrack}^{1/m_{1}}} + \frac{(D)_{2}}{{\left( D_{m} \right)_{2}\left\lbrack {f_{a}/\left( {1 - f_{a}} \right)} \right\rbrack}^{1/m_{2}}}}}\end{matrix}\quad$

Where (Dx)₁ is for (D)₁ “alone” that inhibits a system x %, and (Dx)₂ isfor (D)₂ “alone” that inhibits a system x % whereas in the numerator,(D)₁+(D)₂, “in combination” also inhibit x %. Note that the denominatorsof the last two terms are the expression of MEE. CI-value <0.9 indicatessynergism, 0.9<CI<1.1 indicates additive effects and CI>1.1 indicatesantagonism.

The respective CI values of the tested combinations are depicted inTable 4. Synergy of the anti-CD4 Nanobody 3F11 was observed incombination with the Nanobody 281F12 (anti-CXCR4), with AMD3100(anti-CXCR4), with T-20 (FUZEON®) and with CADA (downregulation of CD4).However, antagonism was observed with the anti-CD4 monoclonal antibodyRPA-T4, suggesting that these compounds may bind to overlapping epitopeson CD4. Synergy of the anti-CXCR4 Nanobody 281F12 was observed with CADAand T-20, but only additive affects were observed with AMD3100 (Table4).

Together these results argue for the combined blockade of HIV1 entry bya bispecific polypeptide (e.g. CXCR4 Nanobody 281F12 and CD4 Nanobody3F11).

Example 5. Construction of Bispecific CXCR4-CD4 Polypeptides

Example 4 demonstrated the synergy effects of the combined blockade ofHIV1 entry by the individual CXCR4 Nanobody 281F12 (SEQ ID NO: 9) andCD4 Nanobody 3F11 (SEQ ID NO: 20). Next, the inventors set out to assessthe effects of dual blockade of both CD4 and CXCR4 receptors inbispecific constructs on HIV infectivity. Bispecific CXCR4-CD4polypeptides were generated as set out below.

Since CXCR4 and CD4 act as co-receptors for gp120, they are expected tobe in close proximity on the cell surface. CCR5, CXCR4, and CD4 arefound predominantly on microvilli on the cell surface, forminghomogeneous microclusters in all cell types, including macrophages andT-cells. Moreover, gp120 induces CD4-CXCR4 membrane colocalization.However, the optimal distance between the two Nanobody building blocksfor simultaneous binding to both receptors and subsequent blockade ofHIV1 entry is not known. For this reason bispecific polypeptides weregenerated with flexible spacers of different lengths for linking the twoNanobody building blocks: (Gly₄SerGly₄) (9GS), (Gly₄Ser)₅ (25GS), and(Gly₄Ser)₇ (35GS), respectively.

Constructs of the anti-CD4 Nanobody 3F11 and anti-CXCR4 Nanobody 281F12were introduced in the production vector pAX100. This vector is derivedfrom pUC119 and contains a LacZ promoter, a kanamycin resistance gene, amultiple cloning site, an OmpA leader sequence, a C-terminal c-myc tagand a His6 tag. Bispecific constructs were generated with 281F12positioned in both N-terminal and C-terminal position of 3F11,genetically fused with the respective linker, yielding 8 differentbispecific constructs (Table 5A). The correct nucleotide sequence of allconstructs was confirmed by sequence analysis (see Table 5B) for anoverview of all sequences). Monovalent and bispecific Nanobodyconstructs were produced in E. coli and purified as myc-His taggedproteins by immobilized metal affinity chromatography (IMAC) usingNickel SEPHAROSE® 6 FF. Nanobodies were eluted from the column with 250mM imidazole and subsequently desalted towards dPBS.

TABLE 5A Panel of CXCR4-CD4 Nanobodies CD4-CXCR4 03F11-9GS-281F1203F11-25GS-281F12 03F11-35GS-281F12 CXCR4-CD4 281F12-9GS-03F11281F12-25GS-03F11 281F12-35GS-03F11

Subsequently, the correct monovalent and bispecific Nanobody constructswere recloned into the pAX205 vector for production in the yeast Pichiapastoris as FLAG3-His6-tagged proteins Plasmids encoding bispecificconstructs were linearized by digestion with restriction enzymes priorto the transformation into P. pastoris strain X-33. Small scale testexpressions of P. pastoris transformants were done in to select for theclone with good expression levels. Hereto 4 ml scale expressions wereperformed of 4 clones of each construct in 24-wells deep well plates.Expression of the Nanobody constructs in the medium was evaluated bySDS-PAGE. Medium fractions were collected and used as starting materialfor immobilized metal affinity chromatography (IMAC) using NickelSEPHAROSE® 6 FF. Nanobody constructs were eluted from the column with250 mM imidazole and subsequently desalted on SEPHADEX® G-25 Superfineon the Atoll (AT0002) towards dPBS. The purity and integrity of Nanobodyconstructs was verified by SDS-PAGE and western blot using anti-VHH andanti-tag detection.

TABLE 5B Name SEQ ID Amino acid sequence 03F11-9GS- 101EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSS 281F12TGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTQVTVSSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGWGPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSS 03F11- 102EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSS 25GS-TGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNP 281F12ARWDGYDFRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGWGPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTY WGQGTQVTVSS 03F11-103 EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSS 35GS-TGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNP 281F12ARWDGYDFRGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGWGPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSS 281F12- 104EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGW 9GS-03F11GPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSSGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTQVTVSS 281F12- 105EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGW 25GS-03F11GPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDF RGQGTQVTVSS281F12- 106 EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGW35GS-03F11 GPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTQVTVSS

Example 6. Binding Analysis of Bispecific CXCR4-CD4 Polypeptides

To assess if the formatting into bispecific constructs affected thebinding of the CXCR4 Nanobody to CXCR4, the entire set of bispecificpolypeptides was analysed for binding to CXCR4 on viral lipoparticles(Integral Molecular). Briefly 2 units of null VLPs and hCXCR4lipoparticles were coated on 96-wells maxisorp plates overnight at 4° C.In the next day free binding sites were blocked using 4% marvel skimmedmilk in PBS for 2 h at room temperature. Then, after washing the plate 3times with PBS, 100 nM, 10 nM, 1 nM and 0 nM of purified polypeptideswere added to the coated wells and incubated for 1 h at roomtemperature. After washing 3 times with PBS, bound polypeptides weredetected with mouse anti-c-myc (Roche, cat#11667149001) and rabbitanti-Mouse-HRP (DAKO, cat#P0260) antibodies both for 1 h at roomtemperature. Binding was determined based on O.D. values and compared tocontrols: an irrelevant Nanobody, a non-coated well, both parentalmonovalent building blocks and a monoclonal anti-CXCR4 antibody 12G5(R&D Systems, cat# MAB170).

FIG. 2.2 shows the results of the binding ELISA to CXCR4 lipoparticlesversus control lipoparticles. An orientation effect for bispecificconstructs with the CD4 Nanobody is observed, and CXCR4 binding was onlyretained with the CXCR4 Nanobody placed at the N-terminal position. Achange in linker length could not overcome this loss of target bindingof the CXCR4 Nanobody, except perhaps for the CD4-25GS-CXCR4 construct,which seemed to be less impaired than the two other bispecifics with theCXCR4 moiety in the C-terminal position.

The panel of CXCR4-CD4 bispecific polypeptides was analysed fordose-dependent binding to cell lines with different relative expressionlevels of the two targets CXCR4 and CD4 in flow cytometry. Cells wereincubated with Fc-blocking solution (Miltenyi Biotec cat#130-059-901)for 30 minutes before staining with monoclonal anti-CXCR4 antibody 12G5(R&D # MAB170) and monoclonal anti-CD4 antibody BA1 (Diaclone#854030000). Bound polypeptides were detected with mouse anti-c-myc (AbDSerotec, cat# MCA2200) and Goat anti-Mouse-PE (Jackson ImmunoResearch,cat#115-115-171) antibodies both for 30 min shaking at 4° C. Binding wasdetermined based on MCF values and compared to controls.

Expression levels of CD4 and CXCR4 on Jurkat cells, THP-1 cells andMolm-13 cells are depicted in FIG. 2.3, as well as the binding curves ofmonovalent and bispecific Nanobody constructs to each of the cell lines.

EC₅₀ values of Jurkat cells and Molm-13 cells are listed in Table 6.Jurkat E6.1 cells show a heterogeneous population of cells expressing noor low levels of CD4. Monovalent CD4 3F11 Nanobody showed only a verylow MCF level of binding to Jurkat cells, although the EC₅₀ value wassimilar to that on THP-1 and MOLM-13 cells (EC₅₀ of 1.1 nM vs 0.5 nM vs0.7 nM, respectively).

TABLE 6 Binding affinity and potency of bispecific CXCR4-CD4polypeptides to cells with different expression levels of CXCR4 and CD4.Functional blockade was assessed by measuring the inhibition of SDF-1mediated chemotaxis via CXCR4. Results are average values of 3experiments. CXCR4⁺/CD4⁺ CXCR4⁺/CD4^(low) MOLM-13 cells Jurkat E6-1cells Inhibition of SDF-1a- Inhibition of SDF-1a- mediated chemotaxismediated chemotaxis Binding IC₅₀ Ratio to Binding IC₅₀ Ratio to IDTarget EC₅₀ (nM) (nM) 281F12 # EC₅₀ (nM) (nM) 281F12 # 281F12 CXCR4 5.286.0 — 7.0 84.2 — 281F12-3F11 CXCR4-CD4 1.1 0.59 146 11 110 0.83F11-281F12 CD4-CXCR4 0.7 1.29 67 1.1 460 0.2 3F11 CD4 0.7 — — 1.4 — — #Fold increase in potency of bispecific relative to monovalent CXCR4Nanobody 281F12.

On Jurkat cells, the bispecific CXCR4-CD4 Nanobody constructs havesimilar EC₅₀ values as the monovalent CXCR4 Nanobody construct, in linewith the high CXCR4 expression levels. The bispecific Nanobodyconstructs have a slightly higher fluorescence level than monovalentCXCR4 Nanobodies.

On double-positive THP-1 cells, a clear shift in the curves of thebispecific CXCR4-CD4 Nanobody constructs is observed compared to bothmonovalent Nanobody constructs. Bispecific Nanobody constructs reachmuch higher plateau MCF levels. The difference in EC₅₀ values betweenbispecifics and monovalent Nanobody constructs however is only moderate(EC₅₀ 0.29 nM (CXCR4-CD4) vs 0.5 nM (CD4) vs 3.1 nM (CXCR4)). On MOLM-13cells the EC₅₀ value of the bispecific polypeptides is similar to thatof CD4 Nanobody 3F11. Also here increased plateau levels are observed.The binding curves of the inverse orientation (CD4-CXCR4) bispecificNanobody constructs overlap with the monovalent CD4 Nanobody 3F11.

This increase in total fluorescence in flow cytometry may representadditive binding (binding to each target alone), as well as simultaneousbinding to both targets on the cell surface.

Example 7: Bispecific Constructs Show Increased Affinity and InhibitoryPotency for CXCR4 7.1: Inhibition of CXCR4-Mediated Chemotaxis byCXCR4-CD4 Bispecific Constructs

To assess if bispecific CXCR4-CD4 polypeptides show increased affinityand potency on cells expressing both receptors, a CXCR4-dependentfunctional assay was performed. Dose-dependent inhibition ofCXCL12-induced chemotaxis by the panel of bispecific CD4-CXCR4Nanobodies was determined on Jurkat (CXCR4⁺/CD4^(low)), and Molm-13cells (CXCR4⁺/CD4⁺⁺) for direct comparison of cells expressing both oronly one receptor.

Bispecific polypeptides were analyzed for inhibition of CXCL12-inducedchemotaxis on cells endogenously expressing CXCR4. As chemoattractant aconcentration of 750 pM SDF-1α (R&D Systems) was used on 100,000cells/well for the Jurkat cell line, and 500,000 cells/well for theMOLM-13 cell line. SDF-1α and serially diluted Nanobody constructs wereadded to the bottom of a small chemotaxis plate (Neuprobe 106-5) in atotal volume of 29 μl. A chemotaxis filter membrane (ChemoTx®Disposibla, pore size 5 μm) was placed on top of the wells, ensuringthat the membrane was in contact with the solution in the wells below. ANanobody dilution (10 μl at 5× the serially diluted final concentrationas below the membrane in each well) was added on top of the membrane,followed by 40 μl of cell suspension. The plates were incubated for 3hours at 37° C. in a humidified incubator (5% CO₂). After incubation,the filters were carefully removed and the cells in the well below wereresuspended in the existing solution. The complete cell suspension wastransferred to the corresponding wells of white polystyrene Costarplates. After this, 30 μl of Cell Titer Glo reagent (Promega G7571) wasadded to each well, followed by a 10 minute incubation, with shaking inthe dark. Luminescence was measured (1 sec/well) using an Envision 2103Multilabel Reader with emission filter 700 (Perkin Elmer). On each platethe corresponding monovalent CXCR4 Nanobody was included as reference,allowing to calculate the fold increase of the bispecific within eachplate. As additional control, 1:1 mixtures of monovalent Nanobodies wereincluded. As reference, anti-CXCR4 antibody 12G5 was included on eachplate.

Results of a representative example are shown in FIG. 2.4, and IC₅₀values are presented in Table 6 (average of n=3 experiments).

The bispecific CXCR4-CD4 constructs showed a strong potency enhancementof about 150-fold on double-positive cells compared to the monovalentCXCR4 Nanobody, whereas the CD4 Nanobody by itself did not have anyeffect on SDF-1 function. Remarkably, bispecific constructs in theinverse orientation were still able to block CXCR4 function, despitetheir strongly reduced affinity for CXCR4 due to the C-terminal positionin the construct, although the blockade was only partial.

Since the functional blockade is mainly mediated via CXCR4, avidity bythe simultaneous binding of the anti-CD4 Nanobody is expected totranslate into increased potency in inhibition of chemotaxis.

This indicates that each of the Nanobodies in the bispecific constructis capable of binding simultaneously to their respective target, andcontributes to avidity on cells that co-express both the receptor andco-receptor.

7.2. Inhibition of CXCL12 to CXCR4 by Bispecific CXCR4-CD4 NanobodyConstructs

The capacity of the monovalent and bispecific CXCR4-CD4 Nanobodyconstructs to displace the natural ligand of CXCR4, SDF-1 or CXCL-12,was assessed in a binding inhibition assay on CD4⁺ T-cells (SUPT-1cells) by flow cytometry. Briefly, human T-lymphoid SupT1-CXCR4 cellswere washed once with assay buffer (Hanks' balanced salt solution with20 mM HEPES buffer and 0.2% bovine serum albumin, pH 7.4) and thenincubated for 15 min at room temperature with the agents diluted inassay buffer at the indicated concentrations. CXCL12^(AF647) (humanCXCL12 carrying an AlexaFluor 647 moiety at its second to last aminoacid position) was obtained from Almac Sciences (Craigavon, UK). Afterthe incubation period with the compounds, CXCL12^(AF647) (25 ng/ml)diluted in assay buffer was added to the cell-compound mixtures andincubated at room temperature for 30 min. Thereafter, the cells werewashed twice in assay buffer, fixed in 1% paraformaldehyde in phosphatebuffered saline (PBS), and analyzed on the FL4 channel of a FACSCaliburflow cytometer equipped with a 635-nm red diode laser (BectonDickinson). The percentages of inhibition of CXCL12^(AF647) binding werecalculated according to the formula(1−[MFI−MFI_(NC)]/[MFI_(PC)−MFI_(NC)])×100 where MFI is the meanfluorescence intensity of the cells incubated with CXCL12^(AF647) in thepresence of the inhibitor, MFI_(NC) is the mean fluorescence intensitymeasured in the negative control (i.e., autofluorescence of unlabeledcells), and MFI_(PC) is the mean fluorescence intensity of the positivecontrol (i.e., cells exposed to CXCL12^(AF647) alone).

The respective IC₅₀ values are shown in Table 7.2.

The anti-CXCR4 Nanobody 281F12 blocked binding of the specificallylabelled CXCL12^(AF647) to CXCR4 expressed with an IC₅₀ of 6.3 nM. Thebispecific constructs 3F11-281F12 and 281F12-3F11 were very similar withpotencies of 1.5 nM and 0.97 nM, respectively, 4 to 6.5-fold betterrelative to monovalent Nanobody 281F12 on T-cells. The anti-CD4 Nanobody3F11 did not interfere with CXCL12^(AF647) binding to CXCR4 on T-cells.

TABLE 7.2 Inhibition of ligand binding and activation of CXCR4 byCXCR4-CD4 polypeptides. SDF-1 induced SDF-1^(AF647)- Ca²⁺ signalingdisplacement U87-CD4- Anti-CXCR4 mAb 12G5 binding inhibition SUPT-1cells CXCR4 SUPT-1 cells THP-1 cells Jurkat cells ID Target IC₅₀ (nM) n= 3 IC₅₀ (nM) n = 3 IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) 3F11 CD4 — — — — —281F12 CXCR4 6.3E−09 6.67E−08 5.13E−08 4.93e−08 1.37e−08 281F12-3F11CXCR4-CD4 9.6E−10 5.31E−09 1.50E−09 2.48e−09 2.93e−08 3F11-281F12CD4-CXCR4 1.5E−09 3.43E−09 5.66E−08 no fit 6.31e−07 AMD3100 CXCR49.3E−08  4.1E−07 1.25E−09

7.3 Inhibition of SDF-1-Induced Calcium Signaling by BispecificCXCR4-CD4 Polypeptide Constructs

The capacity of the monovalent and bispecific CXCR4-CD4 constructs toinhibit down-stream signal transduction of CXCR4 receptor was assessedin inhibiting CXCL-12-induced Ca²⁺-signaling. To this end U87.CD4.CXCR4glioblastoma cells were loaded with the fluorescent calcium indicatorFluo-3 acetoxymethyl (Molecular Probes) at 4 μM in assay buffer (Hanks'balanced salt solution with 20 mM HEPES buffer and 0.2% bovine serumalbumin, pH 7.4) for 45 min at room temperature. After thorough washingwith assay buffer, cells were pre-incubated for 10 min at 37° C. in thesame buffer with the Nanobody constructs or AMD3100 for 10 min at 37° C.Next the intracellular calcium mobilization in response to CXCL12 wasmeasured at 37° C. by monitoring the fluorescence as a function of timesimultaneously in all the wells by using a Fluorometric Imaging PlateReader (FLIPR; Molecular Devices, Sunnyvale, Calif., USA), in essence asdescribed by Princen et al. (Princen et al., 2003 Cytometry 51, 35-45).

The IC₅₀ values are shown in Table 7.2.

None of the monovalent polypeptides induced by themselves anysignificant Ca²⁺-signaling. No differences in IC₅₀ values were observedbetween the different orientations of the bispecific polypeptidesCXCR4-CD4 and CD4-CXCR4 in inhibiting CXCL-12-induced Ca²⁺-signaling inU87.CD4.CXCR4 cells. Potency enhancements relative to monovalentpolypeptide CXCR4 281F12, which had an IC₅₀ of 66.7 nM, were 12 to20-fold. Monovalent Nanobody 3F11 displayed no CXCL-12-inducedCa²⁺-signaling inhibition.

Taken together, these results indicate that the simultaneous binding toboth CD4 and CXCR4 by the bispecific CXCR4-CD4 construct on cells thatco-express both targets enhances the affinity and potency of the CXCR4binding moiety, without clear influence on orientation.

7.4 Inhibition of Anti-CXCR4 Antibody Binding by Bispecific CXCR4-CD4Polypeptide Constructs

The capacity of the monovalent and bispecific CXCR4-CD4 Nanobodyconstructs to displace the binding of anti-CXCR4 mAb 12G5 was assessedon different cell lines, SUPT-1 CD4⁺ T-cells, THP-1, and Jurkat cells byflow cytometry.

In short, cells were washed once with assay buffer (Hanks' balanced saltsolution with 20 mM HEPES buffer and 0.2% bovine serum albumin, pH 7.4)and then incubated for 15 min at room temperature with the Nanobodiesdiluted in assay buffer at the indicated concentrations. Next,anti-CXCR4 mAb 12G5 (PE-labelled, 10 nM) was added to the cell-agentmixtures and incubated at room temperature for 30 min. Thereafter, thecells were washed twice in assay buffer. In case of SUPT-1, cells werefixed in 1% paraformaldehyde in phosphate buffered saline (PBS).Subsequently, the cells were analyzed on the FL2 channel of a FACSCalibur flow cytometer (Becton-Dickinson).

Results are depicted in Table 7.2 and FIG. 3.

On Jurkat cells, the bispecific 281F12-3F11 Nanobody construct lost2-fold potency compared to monovalent CXCR4 Nanobody construct, in theinverse orientation a 50-fold loss was observed. On SUPT-1 and THP-1cells, which co-express CXCR4 and CD4, the bispecific 281F12-3F11Nanobody construct showed enhanced displacement of 12G5 mAb bindingcompared to monovalent 281F12 Nanobody construct albeit with bi-phasiccurves. It was hypothesized that this is due to the avidity provided bythe CD4 binding of the 3F11 Nanobody arm.

Example 8: Potent and Broad HIV1 Neutralization by Bispecific CXCR4-CD4Constructs

The specificity of the inhibitory effects of the bispecific CXCR4-CD4constructs and the corresponding monovalent CXCR4 and CD4 constructswere tested on CXCR4-using (X4) HIV-1 clone NL4.3 infecting MT-4 cells,and in phytohemagglutin (PHA) stimulated PBMCs (expressingCD4/CXCR4/CCR5) from different healthy donors. The CCR5-using (R5) HIV-1strain BaL was used to infect PBMCs.

8.1. HIV-1 Infection Assays

The anti-HIV-1 potencies of the bispecific CD4-CXCR4 Nanobody constructsand the corresponding monovalent CXCR4 and CD4 Nanobody constructs weredetermined by measuring the cytopathic effect of distinct HIV-1 strainsin MT-4 and U87 cell lines, or by quantification of the viral p24antigen production in the culture supernatant of PBMCs, as described inExample 3.2.

The HIV1 neutralisation results in MT-4 cells were depicted as IC₅₀values in Table 8.1.1.

In MT-4 cells infected with the NL4.3 strain, the CXCR4 Nanobodyspecifically inhibited anti-X4 HIV1 entry via CXCR4, but did not inhibitbinding to CCR5. The CD4 Nanobody effectively blocked both X4 HIV1infection, with a similar IC₅₀ value as the CXCR4 monovalent in MT-4cells. Bispecific CXCR4-CD4 constructs were extremely potent ininhibiting HIV-1 X4 virus replication in MT-4 cells in PHA-stimulatedPBMCs, with 30-370 pM potencies. For the bispecific CXCR4-CD4 constructspotency increases were between 250-320 fold compared to the monovalentCXCR4 Nanobody, with the shortest linker seeming to be slightly betterthan the longer linkers. Bispecific polypeptides with Nanobodies in theinverse orientation, i.e. with the reduced affinity towards CXCR4, wereless active in this functional assay, but still considerable more potentthan the CD4 monovalent.

Next, we assessed if the observed potencies of the bispecific constructswere due to the combined blockade by the CXCR4 and CD4 Nanobodies, orwhether linking of the two Nanobodies into the bispecific construct wasnecessary for the potency enhancement. To this end, the inhibition ofNL4.3 infectivity in MT-4 cells was compared for bispecific281F12-35GS-3F11 Nanobody construct, and monovalent Nanobodies eitheralone or in a 1:1 molar ratio.

Results are shown in FIG. 4.

While the mixture of monovalent CXCR4 and CD4 Nanobody constructsresulted in an approximately 2-fold improved IC₅₀ compared to the bestmonovalent Nanobody, the bispecific construct gave a 320-foldimprovement, with 150 pM potency. Thus, the simultaneous binding to bothCXCR4 and CD4 of the bispecific CXCR4-CD4 polypeptides resulted inavidity and strongly enhanced potencies in the neutralization ofCXCR4-using HIV-1 compared to the monovalent counterparts alone or thecombination of the monovalents. The linking is important, but no cleareffect of linker lengths is apparent.

TABLE 8.1.1 Anti-HIV specificity profile of bispecific Nanobodies withdifferent linker lengths for CXCR4-tropic NL4.3 (X4), and CCR5-tropic(R5) BaL viruses. IC₅₀ (nM) Cells + HIV strain MT-4 + U87 + PBMC + NL4.3 NL 4.3 NL 4.3 PBMC + (X4) (X4) (X4) BaL (R5) Nanobody n = 3 n = 3 n= 3 n = 3 CD4 3F11 35.3 >1333 580 #  610 CXCR4 281F12 22.9 >666627.7 >1666 CD4-9GS-CXCR4 14.9 >3333 17.0 >666 CD4-25GS-CXCR4 9.2 >3333 8.7 383.3 CD4-35GS-CXCR4 6.1 >3333 28.9 38.4 CXCR4-9GS-CD4 0.20 0.53 0.03 CXCR4-25GS-CD4 0.21 2.67  0.12 CXCR4-35GS-CD4 0.19 2.67  0.09 2.6AMD3100 4.75 10  4.5 — # High donor variability observed.

The Nanobodies were further evaluated for their anti-HIV activity inPHA-stimulated PBMCs from different donors with additional X4 anddual-tropic X4-R5 specific HIV clones. For these experiments werestricted ourselves to bispecific constructs with the longest linker(35GS; since there was no clear effect of the linker length), along withthe corresponding monovalent Nanobodies and AMD3100.

PHA-stimulated blasts were seeded at 0.5×10⁶ cells per well into a48-well plate (Costar; Elscolab, Kruibeke, Belgium) containing varyingconcentrations of compound in medium containing IL-2. The virus stockswere added at a final dose of 100 TCID50 of HIV-1 or HIV-2. At 8-10 daysafter the start of the infection, viral p24 Ag was detected in theculture supernatant by an enzyme-linked immunosorbent assay (PerkinElmer, Brussels, Belgium). For HIV-2 p27 Ag detection, the INNOTEST fromInnogenetics (Temse, Belgium) was used. In each assay AMD3100 wasevaluated as control compound.

Results are shown in Table 8.1.2.

The anti-CXCR4 Nanobody 281F12 inhibited very consistently HIV-1 NL4.3in every PBMC donor, with an IC₅₀ of 46.7 nM. The anti-CD4 monovalentNanobody 3F11 was weakly active against HIV-1 NL4.3.

In 5 different PBMC donors an IC₅₀ of about 580 nM was obtained, but notin nine other PBMC donors, where no activity was measured (for reasonsthat are currently unclear). The bispecific 281F12-35GS-3F11 constructdisplayed potent anti-HIV-1 activity with an IC₅₀ as low as 86.7 pM (2.6ng/ml), whereas the bispecific 3F11-35GS-281F12 construct consistentlyinhibited replication with average IC₅₀ of 29 nM. AMD3100 had an averageIC₅₀ of 3.3 nM.

The Nanobodies were further evaluated for their anti-HIV activity toX4-R5 dual-tropic HIV isolates.

The dual-tropic (R5/X4) HIV-1 strain HE and the dual-tropic (R5/X4)HIV-2 ROD strain were initially investigated on human MT-4 cells, thatendogenously express CD4 and CXCR4, but not CCR5. The (R5/X4) HIV-1 HEstrain was initially isolated from a patient at the University Hospitalin Leuven. Activity (IC₅₀) and toxicity (CC₅₀) were determined usingmicroscopic evaluation and MTS viability staining method. Consistent pMpotencies were obtained for the most potent bispecific 281F12-3F11construct on the dual-tropic HIV1 HE and HIV2 ROD strains.

In PBMCs, that express both CCR5 and CXCR4 co-receptors, Nanobody 3F11was not active against the dual-tropic R5/X4 HIV-1 HE, while Nanobody281F12 was modestly active with an IC₅₀ of 266.7 nM. In contrast, thebispecific 281F12-35GS-3F11 construct displayed potent anti-HIV-1 HEactivity with an IC₅₀ of 1.5 nM, whereas the bispecific 3F11-35GS-281F12construct lost very often its activity.

The activity of AMD3100 is also variable and lost in the assayssometimes its activity, very likely due to the level of co-receptorexpression of CXCR4 (very high) and CCR5 (very low, but variable 1-20%)on the donor PBMC. Notably, AMD14031/maraviroc never showed anysignificant anti-HIV-1 HE activity in this cell assay system.

Together these results indicate that bispecific polypeptides have abroad coverage in different X4 and dual-tropic X4-R5 HIV strains, andconsistent high potency in the picoMolar-low nanoMolar range in blockingvirus infections.

TABLE 8.1.2 Anti-HIV activity profile of Nanobodies towards distinctdual tropic isolates on MT-4 cells and on PBMCs, in comparison to the X4strain NL4.3. MT-4 PBMC NL4.3 HE HIV-2 ROD NL4.3 HE SM145 DJ259 BaL CellX4 R5/X4 R5/X4 X4 R5/X4 R5 R5 R5 HIV strain n = 3 n = 3 n = 3 n = 10 n =9 n = 2 n = 2 n = 6 Nanobody IC50(M) 3F11 3.47E−08 1.00E−08 2.27E−085.0E−07 >1.7E−06 2.7E−08   3.1E−08   6.1E−07 281F12 2.27E−08 1.00E−088.67E−08 4.7E−08   3.5E−07 nd >1.7E−06 >1.7E−06 281F12-35GS-3F111.87E−10 9.06E−11 3.00E−10 2.9E−10   1.5E−09 1.6E−09   1.5E−09   2.6E−093F11-35GS-281F12 6.00E−09 2.00E−09 8.75E−09 2.3E−08   4.8E−07 7.5E−09  5.5E−08   3.8E−08 AMD3100 4.28E−09 3.90E−09 2.11E−08 3.3E−09   2.6E−08—

8.2 Specificity for CXCR4-Co-Receptor Usage

The potency of the CXCR4 Nanobody is specific for HIV-1 strains thatdepend on CXCR4 co-receptor usage for entry. One potential disadvantageof blockade of only one of the HIV-1 co-receptors is that it may triggerthe re-emergence of the HIV subtype that is not originally targeted.

We tested the HIV activity of bispecific Nanobody constructs on distinctCCR5-dependent HIV-1 strains, (R5) HIV-1 strain BaL (obtained from theMedical Research Council AIDS reagent project (Herts, UK), and theclinical isolates DJ259 (clade C) and SM145 (clade C) on PBMCs ofdifferent donors. In R5 viruses only the CD4 Nanobody in the bispecificconstruct contributes to the virus neutralization. Without being boundto any theory, it was hypothesized that since CXCR4 is expressed onPBMCs, in these cells the CXCR4 Nanobody in the bispecific polypeptidecan bind to CXCR4 and contribute to avidity, and in this manner enhancethe inhibition potency of the CD4 Nanobody.

Results are shown in Tables 8.1.1 and 8.1.2.

Bispecific CXCR4-CD4 constructs can inhibit infectivity of BaL in MT-4cells with an IC₅₀ value of 2.5 nM, around 200-fold enhanced potencyrelative to the potency of monovalent CD4 Nanobody (Table 8.1.1).Bispecific CXCR4-CD4 constructs are more potent inhibitors of BaL thanconstructs in the inverse orientation, probably due to the unfavourableposition of 281F12, in which the CXCR4 binding is impaired. The resultswere confirmed with neutralization of two R5 clinical isolates, SM1145and DJ259, where the bispecific CXCR4-CD4 construct maintained 1.5 nMpotencies, i.e. having 17 and 20 fold better potencies than themonovalent Nanobody 3F11 alone.

Together these results indicate that the cell binding affinity of theCXCR4 Nanobody, even on R5 HIV1 strains where CXCR4 Nanobody it is notactively contributing to functional entry blockade, the CXCR4 Nanobodycontributes to the high potency of the bispecific CXCR4-CD4 polypeptide.

Thus, the bispecific polypeptides of the invention can be effectivelyused to treat an infection in which HIV is resistant against one moietyor uses another co-receptor (e.g. a CR not targeted by the bispecificpolypeptide).

8.3 Neutralization of Entry-Inhibitor Resistant HIV1 Viruses

To substantiate the contribution of an “anchor” in the avidity of theother moiety in the bispecific polypeptide, blockade of HIV infectionwas assessed for a panel of HIV-1 mutant viruses that were maderesistant for the CXCR4 small molecule inhibitor AMD3100, the CXCR-4ligand, or the control antibody 12G2 (Polymun Scientific (Vienna,Austria)).

The IC₅₀ values of the bispecific CXCR4-CD4 Nanobodies towards AMD3100resistant virus are depicted in FIG. 5, and in Table 8.3.

Monovalent CXCR4 Nanobodies showed a 100-fold loss in potency, similaras AMD3100, while the CD4 potency was unaffected. Each of the CXCR4-CD4bispecific polypeptides had retained potencies below 1 nM for blockinginfection of AMD3100 resistant virus, 20-fold better than the monovalentCD4 building block. Over the complete panel of resistant viruses, theCXCR4-CD4 bispecific polypeptide retained strong neutralizing potencywith IC₅₀ values between 0.3-1.1 nM.

Thus, bispecific polypeptides seem relatively insensitive to mutantsthat no longer bind to one of the targets.

8.4 Generation of 3F11 and 281F12 Resistant HIV-1 NL4.3 Viruses

Viral escape mutants were generated by culturing NL4.3 in the presenceof monovalent Nanobodies at IC₉₀ concentration over multiple passages.The HIV-1 NL4.3 3F11-resistant virus was obtained after seven months ofcell culture, passaging MT-4 cells in increasing concentrations of themonovalent Nanobody (starting from EC₅₀ concentrations). The HIV-1 NL4.3281F12-resistant virus was finally obtained in more than 2 years ofdedicated cell culturing passaging HIV-1 NL4.3 in the presence ofNanobody 281F12 directed against CXCR4. For comparison, the generationof resistant AMD3100 strains was obtained after 11 months.

The Env gp120 sequence of the resistant strains were determined,yielding the following mutations:

-   -   gp120 of 3F11-^(res) strain: V40(A,V), R118(K,R), N158(N,S),        S160(N,S), T311(T,I), T378(T,I).    -   gp120 of 281F12^(res) strain: S169L, V170N, M2961, H300Y (V3        region), S435F, K460N, L4641.

Resistant viral clones thus identified were used for testing thepotencies of bispecific polypeptides compared to the monovalentpolypeptides.

Respective IC₅₀ values are presented in Table 8.3.

Nanobody 3F11 (anti-CD4) lost completely its activity against the HIV-1NL4.3 3F11^(res) virus but also against the HIV-1 NL4.3 281F12^(res)virus. However, the blocking capacity of 3F11 is maintained on theSDF-1^(res) and AMD3100^(res) viruses, suggesting that the loss isspecific to the mutations of the 281F12^(res) strain, and not related tothe gp120-CXCR4 interaction per se.

Nanobody 281F12 (anti-CXCR4) completely lost activity against HIV-1NL4.3 281F12^(res) virus and AMD3100^(res) virus, but was active againstthe HIV-1 NL4.3 3F11^(res) virus, with an activity comparable to thewild-type virus (IC₅₀: 0.3 μg/ml) when the virus stocks wereappropriately titrated and re-evaluated in MT-4 cells. AMD3100 almostkept its activity against the HIV-1 NL4.3 3F11^(res) virus (IC₅₀ 18 nM,14 ng/ml), but lost significantly activity against the HIV-1 NL4.3281F12^(res) virus (IC₅₀ 400 nM, 317 ng/ml), suggesting overlappingbinding sites on CXCR4.

TABLE 8.3 Anti-HIV activity profile of bispecific CXCR4-CD4 constructswith entry-inhibitor resistant HIV-1 NL4.3 variants determined in MT-4cells and PBMCs. IC₅₀ (M) Cells MT-4 ID NL4.3 wt 3F11 res. 281F12 res.AMD-3100 res. CXCL-12 res. 2G12 res. 3F11 3.47E−08 >6.7E−06 >6.7E−062.27E−08 1.53E−07 2.33E−08 281F12 2.27E−08 8.73E−08 2.33E−06 >1.7E−062.20E−07 1.73E−08 CXCR4-35GS-CD4 1.87E−10 3.10E−10 1.40E−09 1.13E−094.33E−10 1.10E−10 CD4-35GS-CXCR4 6.00E−09 9.57E−08 >3.1E−07 1.40E−087.00E−08 3.00E−09 AMD3100 4.28E−09 1.85E−08 3.99E−07 4.04E−07 5.03E−08PBMC IC50 (M) NL4.3 WT NL4.3 3F11 res. NL4.3 281F12 res. ID n = 10 n = 2n = 2 3F11 5.0E−07 >6.7E−06 >6.7E−06 281F12 2.8E−08   1.2E−07   3.1E−07CXCR4-35GS-CD4 8.7E−11   1.2E−09   1.9E−09 CD4-35GS-CXCR4 2.9E−08  5.2E−07 >6.7E−06 AMD3100 4.5E−09   2.0E−08   5.7E−08

The bispecific 281F12-35GS-3F11 polypeptide kept full activity againstthe HIV-1 NL4.3 3F11^(res) virus, and remarkably lost only about 8-foldactivity against the HIV-1 NL4.3 281F12^(res) virus (from 187 pM to 1.4nM comparing wild-type and resistant virus), while the potency towardsNL4.3 3F11^(res) virus was almost intact. The bispecific3F11-35GS-281F12 polypeptide, that is largely dependent on CD4 Nanobodybinding for its functionality, lost 16-fold activity against the HIV-1NL4.3 3F11^(res) virus (from 6 nM to 96 nM comparing wild-type andresistant virus) and completely lost its activity against the HIV-1NL4.3 281F12^(res) virus.

These data indicate that even on viruses that are resistant to one ofthe targets, the bispecific CXCR4-CD4 polypeptide retains a strongpotency in the picoMolar range in inhibition of HIV1 entry, suggestingthat functionality of only one of the arms of the bispecific CXCR4-CD4polypeptides is sufficient for the potent inhibition, when the other armcan provide binding avidity. Indeed, we have not yet succeeded ingenerating double resistant HIV.

Together these results indicate that bispecific polypeptides have abroad coverage in different HIV strains (see Table 8.3).

Bispecific polypeptides may thus represent a powerful means to overcomeresistance to HIV1 infection.

8.5 Blockade of HIV1 Infectivity in TZM-Bl Cell-Based Assays

The panel of bispecific CXCR4-CD4 polypeptides and the correspondingmonospecific Nanobodies were also evaluated for their anti-HIV-1activity in TZM-bl cells, i.e. HeLa cells that are expressing low levelsof CXCR4 transfected with human CD4 and CCR5.

TZM-bl cells were seeded in transparent 96-well plates at 1×10⁴ cellsper well in DMEM (Dulbecco's Modified Eagle Medium; Life Technologies,Waltham, Mass., USA) with 10% Fetal Bovine Serum (FBS) and 10 mM HEPES.Subsequently, compounds were added and the cell/compound mixture wasincubated at 37° C. After 30 min, HIV was added at 100 pg p-24 HIV-1Agper well. After 48 h of incubation, the assay plates were analyzed. Forthe analysis, Steadylite plus substrate solution (PerkinElmer, Waltham,Mass., USA) was added to the assay plates. The luminescent signal of thelysed cell suspension was analyzed in white 96-well plates on aSpectraMax L luminescence microplate reader (Molecular Devices,Sunnyvale, Calif., USA) after a 10 min incubation period in the dark.Luciferase activity induced by HIV-1 Tat protein expression was measuredas an assessment of the amount of HIV replication (cf. Measuring HIVneutralization in a luciferase reporter gene assay. Montefiori, MethodsMol Biol. 2009; 485:395-405).

The results are provided in Table 8.5

TABLE 8.5 Anti-HIV activity profile of CXCR4-CD4 polypeptides in TZM-bIcell-based assays. Cells TZM-bI HIV strain R5 X4 SM145 DJ259 NL4.3 WTNL4.3 281F12 res NL4.3 3F11 res ID IC₅₀ (M) n = 2 IC₅₀ (M) n = 2 IC₅₀(M) n = 4 IC₅₀ (M) n = 3 IC₅₀ (M) n = 3 3F11 8.33E−09 1.69E−071.31E−07 >6.7E−06 >6.7E−06 281F12 >6.7E−06 >6.7E−06 2.35E−08 3.66E−074.56E−08 281F12-35GS-3F11 1.19E−08 1.56E−07 2.66E−11 2.52E−11 3.78E−113F11-35GS-281F12 1.41E−08 1.73E−07 1.82E−09  6.8E−08 4.53E−09 AMD31004.11E−07 4.92E−06 9.15E−07 AMD14031 2.82E−06 7.54E−06

In these cells, Nanobody 3F11 (anti-CD4) inhibits X4 HIV-1 NL4.3replication with an IC₅₀ of 131 nM, while the anti-CXCR4 Nanobody 281F12had an IC₅₀ of 23.5 nM. The bispecific polypeptide 281F12-35GS-3F11displayed anti-HIV-1 activity with an IC₅₀ as low as 27 pM, while3F11-35GS-281F12 inhibited X4 HIV-1 NL4.3 in TZM-bl cells with an IC₅₀of 1.8 nM. The potencies were preserved in NL4.3 strains that wereresistant to either the CXCR4 281F12 or the CD4 3F11 Nanobodies.

None of the Nanobodies or bispecific polypeptides, nor AMD3100, wasactive against the dual-tropic R5/X4 HIV-1 HE, the dual-tropic R5/X4HIV-2 ROD and the R5 HIV-1 BaL virus in TZM-bl cells. Of note, thespecific CCR5 inhibitor AMD14031/maraviroc that was used as control inthese assays did not block HIV-1 NL4.3, HIV-1 HE nor HIV-2 ROD, butpotently blocked R5 HIV-1 BaL virus (IC₅₀: 4.2 μM).

On two other R5 clinical isolates, the bispecific CXCR4-CD4 Nanobodyretained nM potency, with both orientations having similar activities.

Example 9: Inhibition of HIV-Mediated Cell-Cell Fusion

During HIV transmission, CD4⁺ T-cells can not only become infected bycell-free virions but, importantly, also by close cell-cell contactswith donor HIV-infected T-cells. To mimic these cell-cell interactionswe co-cultured persistently HIV-1-infected cells (HUT-78/HIV-1) withnon-infected SupT1 CD4⁺ target T-cells. Many syncytia, or giant cells,are formed between infected and uninfected T-cells in less than 20hours. Persistently HIV-1 infected HUT-78 cells were generated byinfection of HUT-78 cells with NL4.3 or HIV1 IIIb. The cells weresubcultured every 3-4 days and persistent virus infection was monitoredin the culture supernatants using HIV-1 p24 Ag ELISA. For theco-cultivation assay, different concentrations of the test compoundsalong with 1×10⁵ SupT1 cells/0.5 mL were added to 96-well plates.HUT-78/HIV-1 cells were thoroughly washed to remove free virus from theculture medium, and 5×10⁴ cells (50 μl) were transferred to the 96-wellplates. After 2 days, the EC₅₀-values were determined microscopically,based on the appearance of giant cells or syncytia in the cellco-cultures. The total number of syncytia was counted.

The respective IC₅₀ values for inhibition of syncytia formation areshown in Table 9.

TABLE 9 Inhibition of HIV-1-mediated cell-cell fusion by bispecificpolypeptides. Co-culture of HIV-1-infected cells (HUT-78/HIV-1 NL4.3 orHIV1 IIIb cells) with non-infected SupT1 CD4⁺ target T cells. Targetcells SupT1 SupT1 Cells/HIV1 strain HUT-78/NL4.3 HUT-78/IIIb NanobodyIC₅₀ (M) n = 4 IC₅₀ (M) n = 3 3F11 2.80E−06 4.6E−06 281F12 3.60E−061.8E−06 281F12-35GS-3F11 2.31E−09 1.1E−09 3F11-35GS-281F12 2.86E−082.3E−08 AMD3100 1.12E−06 2.2E−05

For monovalent 3F11, the average IC₅₀ value was 2.7 μM and 3.6 μM for281F12. Bispecific polypeptide 281F12-35GS-3F11 blocked potently with anIC₅₀ of 2.3 nM, while the bispecific polypeptide 3F11-35GS-281F12 had anIC₅₀ value of 28.7 nM. AMD3100 lost activity in this cell-celltransmission assay, compared to its activity in HIV replication assays,displaying an IC₅₀ of 1.1 μM. Thus, bispecific polypeptides are the mostpotent compounds in interfering with the HIV cellular (co-)receptor/gp120-mediated fusion processes.

Example 10: CXCR4 Nanobodies Binding to the Gp120 Binding Site

We further pursued CXCR4 Nanobodies which specifically block HIV entryand preferably do not interfere with natural CXCR4 signal transduction.

In order to identify CXCR4 Nanobodies that block specifically theinteraction of gp120 on CXCR4, but which do not or minimally interferewith CXCL12 binding, a panel of 70 previously identified CXCR4Nanobodies was analysed for their ability to neutralize infection ofNL4.3 HIV1 in MT-4 cells. CXCR4 specific Nanobodies were also evaluatedin PBMC isolated from buffy coats of blood from healthy donors andtested against X4 HIV-1 NL4.3 and R5 HIV-1 BaL replication. IC₅₀ valuesof neutralization of MT-4 cells are depicted in FIG. 6, indicating arange of potencies, with the most potent Nanobody 15A01 in thesub-nanoMolar range. None of the CXCR4 Nanobodies neutralized theinfection of BaL R5 in PBMCs, as expected (data not shown).

In addition, CXCR4 Nanobodies were analysed for ligand competition bydisplacement of biotinylated SDF-1 on transient transfected Caki cellsexpressing hCXCR4 in flow cytometry. To this end, serial dilutions ofNanobodies were pre-incubated with 30 nM of biotinylated SDF-1 (R&DSystems Fluorikine kit) and incubated to Caki-CXCR4 cells for 1 hour at4C, after which ligand binding was visualised using extravidin-PE. Thebiotin-SDF-1 competitor concentration used in this assay was below theEC₅₀ value obtained in dose-titration, where IC₅₀ values should reflectthe Ki. Ligand displacement IC₅₀ values were calculated, and compared tothe NL4.3 (X4) neutralization potencies.

Comparing the different potencies, several CXCR4 Nanobodies had a largerthan 10-fold difference between IC₅₀ value in HIV1 neutralisationcompared to ligand displacement, as depicted in FIG. 7. CXCR4 Nanobodies15F5 and 15G11 were of particular interest, showing hardly any liganddisplacement to CXCR4. Both Nanobodies have substantial HIV1neutralisation capacity, with better potencies than 281F12 Nanobody(IC₅₀ values of 4.7 nM and 17.7 nM, respectively).

Hence, a panel of Nanobodies was generated having a range of potenciesin HIV blocking as well as in ligand displacement.

Example 11: Characterisation of Gp120-Competing CXCR4 Nanobodies

The monovalent CXCR4 Nanobodies that showed binding to the gp120 bindingsite (15F5, 15611, 10C3 and 15A1) were further characterized withrespect to binding to human and cynomolgus CXCR4, as well as to humanCXCR4 variants with defined point mutations in extracellular loop 2 thatwere previously described (Jaenchen et al. 2011).

CXCR4 Nanobodies were allowed to bind to HEK cells transfected withhuman CXCR4, cynomolgus (“cyno”) CXCR4, hCXCR4-V196E, hCXCR4 D187V, andhCXCR4 F189V, respectively. The anti-CXCR4 mAb 12G5 was binding to allpoint-mutants and thus served as a control for membrane expression(Jaenchen et al. 2011). For the epitope mapping, transient transfectionsof the CXCR4 mutants, cyno CXCR4 and wildtype human CXCR4 in thepCDNA3.1 vector were done in HEK293T cells, after which Nanobody bindingwas assessed by flow cytometry using detection of the Myc-tag, followedby secondary anti-mouse PE. Two concentrations of Nanobody were tested,10 nM and 100 nM. The experiment was repeated with essentially the sameresults. Binding of the Nanobodies to HEK293T hCXCR4 cells was used fornormalization using the following formulas. The percentages of Nanobodybinding to the respective mutant receptors were calculated according tothe formula:(1−[(MFI_(hCXCR4)*ratio_(12G5 mAb))−MFI_(mutant)]/[MFI_(hCXCR4)*ratio_(12G5 mAb)])×100,where MFI is the mean fluorescence intensity of the anti-myc detection,and ratio 12G5 mAb: (MFI 12G5_(mutant)/MFI 12G5_(hCXCR4)). Percentage ofbinding to the mutant receptors was calculated for each Nanobodyconcentration, and a position was considered as critical when less than25% residual binding was observed.

Results of the CXCR4 binding analysis are depicted in Table 11.

CXCR4 Nanobodies 15F5 and 15A1 bind equally well to cyno CXCR4 as tohuman CXCR4, and are only sensitive to mutation of residue F189V.Binding of Nanobody 15G11 is impaired by mutations at positions F189V,V196E and D187Vin the extracellular loop 2, while it binds well to cynoCXCR4. Binding of Nanobody 10C3 is reduced on all tested CXCR4 mutantsas well as on cyno CXCR4.

TABLE 11 Binding analysis of CXCR4 Nanobodies to mutant CXCR4 receptorsexpressed on Hek293T cells. % binding # cyno CXCR4 hCXCR4-V196E hCXCR4D187V hCXCR4 F189V CXCR4 Nb 100 nM 10 nM 100 nM 10 nM 100 nM 10 nM 100nM 10 nM 10C3 51.3 57.5 0.6 0.4 24.7 12.8 0.6 0.9 15F5 81.1 101.9 77.693.3 117.1 126.2 8.8 1.5 15G11 96.1 95.1 39.3 22.1 50.3 35.4 0.1 0.115A1 78.9 107.3 77.3 71.3 107.5 115.0 1.9 0.8 281D4 85.9 97.0 79.5 70.50.6 0.9 0.5 0.5 12G5 mAb 100 100 100 100 100 100 100 100 # % binding tocynomolgus (cyno) or mutant CXCR4 receptors expressed on HEK cells,relative to hCXCR4 binding. Expression levels were normalised to 12G5binding.

CXCR4 Nanobodies were further characterized for inhibition of binding ofCXCR4 antagonists to CXCR4. For competition experiments, serialdilutions of CXCR4 Nanobodies were pre-incubated with 1 nM of anti-CXCR4antibody 12G5, and allowed to bind to Jurkat cells. Briefly serialdilutions the different Nanobodies ranging from 500 nM to 0.05 nM wereincubated with 1 nM of 12G5 for 1 h at RT. Then, this mix was added andincubated with the cells for 30 min shaking at 4° C. After washing 3×with PBS 10% FBS, bound antibody (12G5) was detected with Goatanti-Mouse-PE (Jackson Immuno-research, cat#115-115-164) for 30 minshaking at 4° C. Inhibition potency is determined based on the decreaseof signal from 12G5 binding in the absence of Nanobody and the signalwhen in the presence of different amounts of Nanobody.

These results are shown on FIG. 8.

All Nanobodies are able to fully displace binding of 12G5 from CXCR4,with monovalent anti-CXCR4 Nanobody 15F5 being the best competitor withIC₅₀ of 1.25 nM, followed by 15G11 (6.2 nM) and 281F12 (13 nM).

To assess competition with anti-CXCR4 AMD3100, a fixed concentration ofCXCR4 Nanobodies at their respective EC₃₀ binding concentration waspre-incubated with serial dilutions of AMD-3100 ranging from 10 000 nMto 1 nM, and allowed to bind to Jurkat cells for 1 h at RT. In parallel1×10⁵ cells were incubated with Fc-blocking solution (Miltenyi Bioteccat#130-059-901) for 30 minutes shaking at 4° C., after which theAMD-3100-Nanobody mix was added and incubated for additional 30 minutesat 4° C. After washing 3 times with PBS 10% FBS, bound Nanobodies weredetected with mouse anti-c-myc (AbD Serotec, cat# MCA2200) and Goatanti-Mouse-PE (Jackson Immunoreseach, cat#115-115-164) antibodies.Inhibition potency is determined based on the decrease of signal when noADM3100 is present and the signal when in the presence of increasingconcentration of this molecule.

These results are shown on FIG. 8.

AMD3100 can fully compete with the binding of all tested CXCR4Nanobodies with a potency of 100 nM.

In conclusion, CXCR4 Nanobodies 15F5 and 15G11 are potent HIV1antagonists, inhibiting with AMD3100 and mAb 12G5 for binding tocell-expressed CXCR4, but are not competing with the CXCR4 ligandCXCL12, and hence are suitable candidates for formatting into bispecificconstructs with anti-CD4 Nanobody 3F11.

Example 12: Generation of Half-Life Extended CXCR4-CD4 BispecificPolypeptides

Bispecific CXCR4-CD4 constructs were formatted with an anti-AlbuminNanobody, in order to extend its half-life in serum for in vivoexperiments. To this end the respective CXCR4 Nanobody was fused to ananti-Albumin Nanobody with a flexible 15GS-linker, followed by the CD4Nanobody linked with a second 15GS linker. CXCR4 Nanobodies 281F12,15F05 and 15G11 were formatted to half-life extended bispecificconstructs (SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109). Asreference monovalent CXCR4 281F12 was also fused to the anti-AlbuminNanobody (SEQ ID NO: 110). The multivalent constructs were generated bymeans of separate PCR reactions (1 for the N-terminal, 1 for the middleand 1 for the C-terminal Nanobody subunit) using different sets ofprimers encompassing specific restriction sites.

All constructs were cloned into a Pichia pastoris expression vectorderived from pPICZa (Life Technologies) and contains the AOX1 promoter,a resistance gene for Zeocin, the necessary replication origins for bothE. coli and P. pastoris and a multiple cloning site preceded by thecoding information for the S. cerevisiae α-MF signal peptide. In framewith the Nanobody coding sequence, the vector codes for a C-terminal(Flag)₃ tag and a (His)₆ tag. The signal peptide directs the expressedNanobodies to the extracellular environment via the secretory pathway ofthe eukaryotic host. After sequence confirmation, the pAX159-derivedexpression constructs were then transformed into P. pastoris X-33according to standard procedures (EasySelect™ Pichia Expression KitManual, Life Technologies). The purification of Nanobodies from theculture medium was done using standard affinity chromatography on theHis-tag, followed by a gel filtration step. The integrity and the purityof all Nanobodies was confirmed by MS analysis and SDS-PAGE. The aminoacid sequences are provided in Table 12.

Example 13: Inhibition of CXCR4-Mediated Chemotaxis by Half-LifeExtended CXCR4-CD4 Bispecifics

To verify the functionality of the half-life extended bispecificCXCR4-CD4 polypeptides, a CXCR4-dependent functional assay wasperformed, essentially as described in Example 7.1. Dose-dependentinhibition of CXCL12-induced chemotaxis by the half-life extendedmonovalent 281F12-ALB and bispecific 281F12-ALB-3F11 was determined incomparison to the same construct without the anti-Albumin building blockon Jurkat E6 (CXCR4+/CD4 low), and Molm-13 cells (CXCR4++/CD4++). Aschemoattractant a concentration of 750 μM SDF-1α (R&D Systems) was usedon 100,000 cells/well for the Jurkat cell line, and 1 nM SDF-1α on500,000 cells/well for the MOLM-13 cell line.

Results of representative experiments are shown in FIG. 9A+B.

The fusion with an anti-Albumin Nanobody did not substantially affectthe affinity and potency of monovalent CXCR4 281F12 or bispecificCXCR4-ALB-CD4 constructs on Jurkat cells, with all Nanobody formatsshowing similar potencies (IC₅₀ values between 16 and 80 nM, dependingon the assay; FIG. 9A). On double-positive MOLM-13 cells, the potency ofthe half-life extended bispecific CXCR4-CD4 constructs was similar tothe corresponding non half-life extended bispecific counterparts,indicating that the anti-Albumin Nanobody did not affect thesimultaneous binding to each of the targets (FIG. 9B).

These results indicate that also the half-life extended bispecificCXCR4-ALB-CD4 constructs are capable of binding simultaneously to theirrespective targets, and that the potency enhancement in blocking CXCR4function relative to the monovalent CXCR4-ALB is maintained.

TABLE 12 Name ID* amino acid sequence 15G11(Q108L)- 107EVQLVESGGGLVQAGDSLRVSCAASGRTSSYAMAWFRQAPGKEREFVGTISRT 15GS-ALB11-NSRTKYADFVEGRFTISRDNAKSTLSLQMTSLKPEDTAVYYCAAKWTGNSYHD 15GS-YTWSKVDEYNVWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGN 03F11(Q108L)SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTLVTVSS 15F05(Q108L)- 108EVQLVESGGGLVRAGDSLRLSCAASGRAFSRYAMGWFRQALGKERELVAAIGW 15GS-ALB11-SPTHTYYADSVKGRFTMSRDNGKNTVFLQMNSLNPEDTAVYYCAAKYSSRDAA 15GS-YRSDYDYNYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSL 03F11(Q108L)RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTLVTVSS 281F12(Q108L) 109EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGW -15GS-ALB11-GPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDST 15GS-WSRSEMYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSL 03F11(Q108L)RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFVAAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAADTYNSNPARWDGYDFRGQGTLVTVSS 281F12(Q108L) 110EVQLVESGGGLVQAGDSLRLSCAASGRAFSRYAMGWFRQAPGKEREFVAAIGW -15GS-ALB11GPSKTNYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYSCAAKFVNTDSTWSRSEMYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS *“ID” denotes SEQ ID NO:

Example 14: Blockade of HIV1 Neutralization by Half-Life ExtendedCXCR4-CD4 Bispecifics

The capacity of the half-life extended bispecific CXCR4-CD4 polypeptidesto block the replication of the CXCR4-using HIV1 strain NL4.3 wasassessed in HIV1 infection assays in the MT-4 cell line, similar asdescribed in Example 8. In this assay, the half-life extended bispecificCXCR4-ALB-CD4 constructs generated with CXCR4 Nanobodies 281F12 and with15F05 were directly compared with the corresponding monovalentNanobodies and the 281F12-35GS-3F11 bispecific polypeptide. AMD3100 wasincluded as reference.

Results are depicted in Table 14.1.

The results indicate that both of the half-life extended CXCR4-ALB-CD4constructs maintained sub nanomolar potency in the HIV1 neutralizationassay on the prototype X4 strain NL4.3, with the construct with CXCR4Nanobody 15F05 having a slightly better potency than the construct with281F12.

These results confirm that the potent anti-HIV1 activity is preserved inhalf-life extended CXCR4-ALB-CD4 Nanobodies.

TABLE 14.1 Inhibition of HIV1 infectivity by half-life extendedCXCR4-CD4 polypeptides of X4 NL4.3 strain in MT-4 cells. AMD-3100 wasused as control compound. Average IC₅₀ of three experiments is shown.Compound Format IC₅₀ (nM) AMD3100 control 6.26 3F11 CD4 Nb 60.69 281F12CXCR4 59.38 281F12-3F11 CXCR4-CD4 0.13 281F12-ALB-3F11 CXCR4-Alb-CD40.33 15F05-ALB-3F11 CXCR4-Alb-CD4 0.23 15F05 CXCR4 NB 4.32

Example 15: Binding of 3F11 to Cynomolgus CD4

For conducting in vivo studies the cross-reactive binding to monkeyorthologues is preferred. For the CXCR4 Nanobodies and the ALB buildingblock in the bispecific CXCR4-ALB-CD4 constructs the cynocross-reactivity was confirmed. To address the cross-reactivity of theanti-CD4 Nanobody, the dose-dependent binding of monovalent CD4 3F11 tothe cynomolgus CD4⁺ HSC-F T cell line was assessed via flow cytometryusing detection of the flag tag (mouse-anti Flag, Sigma cat nr. F1804).

The results are shown in FIG. 10.

The results indicate that 3F11 is cross-reactive to cynomolgus CD4, butthat no saturation is reached, and that the binding affinity tocynomolgus CD4 is reduced compared to human CD4.

1. Polypeptide comprising a first and a second immunoglobulin singlevariable domain (ISV), wherein said first ISV binds to CD4 present onthe surface of a cell; said second ISV binds to CXCR4 present on thesurface of said cell; and wherein said first ISV essentially consists of4 framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which (i) CDR1 ischosen from the group consisting of SEQ ID NOs: 85, 84, 83 and 82; andamino acid sequences that have 1, 2 or 3 amino acid difference(s) withSEQ ID NOs: 85, 84, 83 and 82; (ii) CDR2 is chosen from the groupconsisting of SEQ ID NOs: 91, 90, 89 and 88; and amino acid sequencesthat have 1, 2 or 3 amino acid difference(s) with SEQ ID NOs: 91, 90, 89and 88; and (iii) CDR3 is chosen from the group consisting of SEQ ID NO:99, 98, 97 and 96; and amino acid sequences that have 1, 2, 3 or 4 aminoacid difference(s) with SEQ ID NOs: 99, 98, 97 and 96; and, wherein saidsecond ISV essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which (i) CDR1 is chosen from the group consisting ofSEQ ID NOs: 35, 34, 36-40; and amino acid sequences that have 1, 2 or 3amino acid difference(s) with SEQ ID NOs: 35, 34, 36-40; (ii) CDR2 ischosen from the group consisting of SEQ ID NOs: 50, 48-49 and 51-56; andamino acid sequences that have 1, 2 or 3 amino acid difference(s) withSEQ ID NOs: 50, 48-49 and 51-56; and (iii) CDR3 is chosen from the groupconsisting of SEQ ID NO: 69, 67-68, 70-75 and amino acid sequences thathave 1, 2, 3 or 4 amino acid difference(s) with SEQ ID NOs: 69, 67-68,70-75.
 2. The polypeptide according to claim 1, wherein said first ISVis chosen from the group consisting of 03F11 (SEQ ID NO: 20), 01B6 (SEQID NO: 17), 01E2 (SEQ ID NO: 18), and 01H12 (SEQ ID NO: 19), and whereinsaid second ISV is chosen from the group consisting of 281F12 (SEQ IDNO: 9), 238D4 (SEQ ID NO: 4), 281A5 (SEQ ID NO: 5), 281E10 (SEQ ID NO:6), 281D4 (SEQ ID NO: 7), 281A6 (SEQ ID NO: 8), 283B6 (SEQ ID NO: 10),283E2 (SEQ ID NO: 11), 283F1 (SEQ ID NO: 12), 15F5 (SEQ ID NO: 13),15G11 (SEQ ID NO: 14), 15A1 (SEQ ID NO: 15) and 10C3 (SEQ ID NO: 16). 3.The polypeptide according to claim 1, wherein said polypeptide inhibitsinfection of human immunodeficiency virus (HIV) or simianimmunodeficiency virus (SIV).
 4. The polypeptide according to claim 3,wherein said HIV is chosen from the group consisting of HIV-1 and HIV-2.5. The polypeptide according to claim 1, wherein said cell is a humancell, preferably a human CD4⁺-cell, even more preferably a human CD4⁺T-cell.
 6. The polypeptide according to claim 3, wherein saidpolypeptide prevents infection of said HIV for at least 3 months, suchas at least 6 months, or even longer such as e.g. 9 m, 11 m, 1 y, 1.5 y,2 y or even longer.
 7. The polypeptide according to claim 3, whereinsaid polypeptide inhibits HIV infection by about 10%, 20%, 30%, 40%,50%, 60%, 80%, 90% and preferably 95% or more, such as 100%, forinstance as measured in a HIV infection assay.
 8. The polypeptideaccording to claim 3, wherein said polypeptide inhibits HIV fusion withCD4⁺CXCR4⁺ cells.
 9. The polypeptide according to claim 1, wherein saidpolypeptide inhibits binding of a natural ligand to said CXCR4 by lessthan about 50%, such as 40%, 30%, or 20% or even less than 10%, such asless than 5%, wherein the natural ligand is Stromal Cell-DerivedFactor-1 beta (SDF-1β) or Stromal Cell-Derived Factor-1 alpha (SDF-1α).10. The polypeptide according to claim 1, further comprising a serumprotein binding moiety or PEG.
 11. The polypeptide according to claim10, wherein said serum protein binding moiety is an immunoglobulinsingle variable domain binding serum albumin.
 12. Composition comprisinga polypeptide according to claim
 1. 13. A method for treating and/orpreventing HIV infection in a human subject comprising administering tothe human subject a polypeptide according to claim 1 in an amounteffective to treat the acquired immune deficiency syndrome in the humansubject.
 14. The method according to claim 13, wherein said polypeptideprevents HIV infection for at least 3 months, such as at least 6 months,or even longer such as e.g. 9 m, 11 m, 1 y, 1.5 y, 2 y or even longer.15. The method according to claim 13, wherein said polypeptide inhibitsHIV infection by about 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90% andpreferably 95% or more, such as 100%, for instance as measured in a HIVinfection assay.
 16. The method according to claim 13, wherein saidpolypeptide inhibits HIV fusion with CD4+CXCR4+ cells.
 17. The methodaccording to claim 13, wherein said polypeptide inhibits binding of anatural ligand to said CXCR4 by less than about 50%, such as 40%, 30%,or 20% or even less than 10%, such as less than 5%, wherein the naturalligand is Stromal Cell-Derived Factor-1 beta (SDF-1β) or StromalCell-Derived Factor-1 alpha (SDF-1α).